Guido Krupp

Telomerase activity in `immortal' fish1

Eukaryotic chromosome termini consist of telomeres, short sequence repeats. According to the telomere hypothesis, DNA replication leads to telomere shortening, resulting in a cellular mitotic clock. Telomerase resets it by telomere synthesis. In mammals with a limited growth phase, telomerase activity in somatic tissues is restricted to stem cell derivatives with high proliferation potential. But other animals, like some fish, grow throughout their life with little senescence. All somatic cells require a high proliferation capacity and telomerase should be active in all cells, irrespective of fish age. Indeed, we detected high telomerase activities in all analyzed organs of rainbow trout (Oncorhynchus mykiss).

Longevity of lobsters is linked to ubiquitous telomerase expression

Mammals have high growth rates in embryonic and juvenile phases and no growth in adult and senescent phases. We analyzed telomerase activity in a fundamentally different animal which grows indeterminately. Lobsters (Homarus americanus) grow throughout their life and the occurrence of senescence is slow. A modified TRAP assay was developed and the lobster telomeric repeat sequence TTAGG was determined. We detected telomerase activities which were dependent on RNA and protein components, required dGTP, dATP and dTTP, but not dCTP. Telomerase products with a five nucleotide periodicity were generated. High telomerase activities were detected in all lobster organs. We conclude that telomerase activation is a conserved mechanism for maintaining long‐term cell proliferation capacity and preventing senescence, not only in cellular models or embryonic life stages but also in adult multicellular organisms.

Telomerase Activity in Melanocytic Lesions : A Potential Marker of Tumor Biology

Telomerase activation, being a cardinal requirement for immortalization, is a crucial step in the development of malignancy. With a view toward diagnostic and biological aspects in melanocytic neoplasia, we investigated the relative levels of telomerase activity in 72 nevi and 16 malignant melanomas by means of a modified telomeric repeat amplification protocol (TRAP) assay, including an internal amplification standard. We further compared telomerase activity with the expression of two different proliferation-specific proteins, Ki-67 and repp86, a protein expressed exclusively in the cell cycle phases S, G2, and M. Telomerase activity was associated with the overall growth fraction (Ki-67) but showed a closer correlation with the expression of repp86. Both telomerase activity and proliferation indices discriminated clearly between malignant melanomas and nevi, but not between common and dysplastic nevi. Nonetheless, a portion of nevi exhibited markedly elevated telomerase activity levels without proportionally increased proliferation. This was independent of discernible morphological changes. Clinicopathological correlations showed an association between high telomerase activity and early metastatic spread in melanomas, linking telomerase to tumor biology. Our results provide arguments in favor of an occasional progression from nevi to melanomas and imply that proliferation measurements in combination with telomerase assays may help to elicit early malignant transformation that is undetectable by conventional morphology.

Development of Diagnostic Oligonucleotide Probes for Four Lactobacillus Species Occurring in the Intestinal Tract

Summary Partial sequences of the 16S rRNA from Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus ruminis were used to design diagnostic species-specific DNA oligonucleotide probes. The specificity of the probes was tested against crude rRNA of 30 strains from 12 Lactobacillus species and 18 Gram-positive representatives from various genera. The detection limit of rRNA from homologous strains in mixed cultures (108 cells of a heterologous species) and in 0.6 g minced beef meat corresponded to 3 × 104 cells, and 1 × 105 cells respectively. A rapid lysis technique of cells was developed that facilitates the isolation of nucleic acids directly in 1.5 ml screw-top vials.

Molecular differentiation of bacteria by PCR amplification of the 16S–23S rRNA spacer

In general, operons for prokaryotic rRNA genes contain a transcribed spacer between 16S and 23S rRNA genes. The length and sequence of this spacer are expected to be highly variable. Polymerase chain reaction (PCR) permits the direct amplification of spacer DNA from small amounts of genomic DNA. Data for a wide range of microorganisms were accumulated, and the spacer lengths varied between 280 and 1300 bp. A further differentiation was achieved by a high-resolution gel electrophoresis method, single-strand conformation polymorphism (SSCP). It was possible to differentiate 15 Mycoplasma species, to analyze mixed samples and to distinguish Streptococcus strains and Clostridium botulinum serotypes. Spacer analysis is a promising method for the differentiation of diverse and closely related species, and also useful in analysis of mixed samples.

Regulation of telomerase activity by alternate splicing of human telomerase reverse transcriptase mRNA in a subset of neuroblastomas

It has been proposed that the regulation of telomerase takes place at the transcriptional level, the expression of the catalytic subunit human telomerase reverse transcriptase (hTERT) being crucial for telomerase activity (TA). Recently, differential splicing of hTERT mRNA has been demonstrated in various tissues during embryonal development, and it has been suggested that only full-length transcripts translate into functionally active telomerase. With this in view, we analyzed the different hTERT transcripts by reverse transcriptase-polymerase chain reaction in neuroblastic tumors and compared the results with the TA, the tumor growth fraction, and the MYCN status. In a series of 38 neuroblastic tumors, high TA and full-length hTERT transcripts were found in nine samples, whereas nine samples showed absence of both enzymatic activity and hTERT transcripts. Interestingly, in another eight samples, low or absent TA coincided with a lack of full-length hTERT transcripts. Eleven samples contained hTERT transcripts with low or undetectable TA and one sample had low TA but no hTERT transcripts. TA correlated with MYCN amplification and was weakly associated with the proliferative activity. Moreover, a significant correlation with tumor progression was observed. Our findings point at a posttranscriptional regulation of TA in a subset of neuroblastic tumors. Because high TA was detected only in tumors with full-length hTERT transcripts, reverse transcriptase-polymerase chain reaction analysis of archival neuroblastic tumor samples might help to appraise the malignant potential in individual cases.

Cell proliferation, carcinogenesis and diverse mechanisms of telomerase regulation

Abstract. Replication of linear genomes is incomplete and leaves terminal gaps. Solutions to this ‘end replication’ problem can be traced back to the prebiotic RNA world: ‘fossils’ of the presumptive archetypes of telomere structure and of the telomerase enzyme are retained in the terminal structures of some RNA viruses. Telomerase expression in mammals is ubiquitous in embryonic tissues but downregulated in somatic tissues of adults. Exceptions are regenerative tissues and, notably, tumor cells. Telomerase activation is controlled by cellular proliferation, and it is an early step in the development of many tumors. In contrast to mammals, indeterminately growing multicellular organisms, such as fish and crustaceae, maintain telomerase competence in all somatic tissues. In human tumor diagnostics, detection of proliferation markers with monoclonal antibodies is well established, and in this review, the significance of additional telomerase assays is evaluated. Telomerase inhibitors are attractive goals for application in tumor therapy, and telomerase knockout mice have proven that telomere erosion limits the lifespan of cells in vivo. In contrast, telomerase stimulation can be used to expand the potential of cellular proliferation in vitro, with possible applications for transplantation of in vitro expanded human cells, for immortalizing primary human cells as improved tissue models and for the isolation of otherwise intractable products, such as genuine human monoclonal antibodies.

Rapid kinetic characterization of hammerhead ribozymes by real-time monitoring of fluorescence resonance energy transfer (FRET).

In established methods for analyzing ribozyme kinetics, radiolabeled RNA substrates are primarily used. Each data point requires the cumbersome sampling, gel electrophoretic separation, and quantitation of reaction products, apart from the continuous loss of substrate by radioactive decay. We have used stable, double fluorescent end-labeled RNA substrates. Fluorescence of one fluorophore is quenched by intramolecular energy transfer (FRET). Upon substrate cleavage, both dyes become separated in two RNA products and fluorescence is restored. This can be followed in real time and ribozyme reactions can be analyzed under multiple (substrate excess) and under single (ribozyme excess) turnover conditions. A detailed comparison of unlabeled, single, and double fluorescent-labeled RNAs revealed moderate kinetic differences. Results with two systems, hammerhead ribozymes in I/II (small ribozyme, large substrate) and in I/III format (large ribozyme, small substrate), are reported.

Functional disruption of IEX-1 expression by concatemeric hammerhead ribozymes alters growth properties of 293 cells

The early response gene IEX‐1 modulates apoptosis and cell growth in a poorly defined fashion. Here, we describe the effect of hammerhead ribozymes specifically disrupting IEX‐1 expression in 293 cells. Compared to vector control, 293 cells exhibit a reduced growth rate and a slowed cell cycle progression, when stably transfected with a concatemeric ribozyme construct. In addition, these 293 cells were much less sensitive to apoptosis induced by an activating Fas/CD95 antibody or by the anti‐cancer drugs etoposide and doxorubicin. By modulating the cell cycle, IEX‐1 might be part of a growth signal if favourable growth conditions prevail, whereas under unfavourable conditions, i.e. death receptor activation, IEX‐1 facilitates apoptosis.

Regulation of telomerase activity in quiescent immortalized human cells

Telomerase is a key enzyme in carcinogenesis; telomerase activity has been found in more than 90% of human tumors. Understanding the regulation of this enzyme will improve our knowledge of tumor biology and may lead to novel strategies in cancer therapy. We examined effects of growth arrest on telomerase activity in the human immortalized cell lines U 937 (lymphoma) and L 428 (Hodgkins disease). Cells were starved by serum depletion for 4 days. After readdition of serum, a recovery phase followed. Cell proliferation was monitored with the monoclonal antibody Ki-S5. In the absence of serum, telomerase levels declined fivefold. After serum readdition, recovery to threefold increased level was observed. Furthermore, the prevalence of telomerase-positive cells in normal tissues is an important issue for understanding tumorigenesis. Our TRAP assay is robust against false positives and in mixed cell samples, we found a rather limited sensitivity of the telomere repeat amplification protocol (TRAP) assay. This means that adequate screenings for telomerase-positive somatic cells have to include enrichment steps.