Introduction

Telomerase, a specialized RNA-directed DNA polymerase that elongates eukaryotic telomeres, is present in 80-85% of lung cancer tissues, but not in normal tissue, making it a good potential target for cancer therapy.^5,9 Lung cancers are the deadliest, having no known successful therapeutic strategy. Since the development of the Telomeric Repeat Amplification Protocol (TRAP) assay in the early 1990s, association of telomerase activity with human cancer has increased dramatically.

Essentially, elevated telomerase activity is indicative of a highly aggressive malignancy and is a probable indication of imminent cancerous invasion.⁸ It has recently been hypothesized that the bioactive lipid ceramide might be active in regulating telomerase activity through downstream signaling pathways of c-myc and PPA2, and the subsequent regulation of telomerase.⁹

Background and Significance

The human telomeric repeat sequence has been identified for more than a decade, but exactly how the mitotic clock keeps time continues to elude contemporary science. In order for a cell to divide, the double stranded DNA in its chromosomes must replicate on an RNA template. The terminal 3’ end of each strand, however, is not duplicated in its entirety by the normal replication mechanism.³ Approximately 1000 short base sequences in length, a telomere, or "end part" of the chromosome, protects this 3’ end with multiple repeats of the TTAGGG hexanucleotide sequence which serves as a molecular cap.² As a cell proceeds through life-cycle divisions the telomeres are progressively shortened, eroding their protective function. Once degradation of the sequence is significant a cell is unable to divide and is "tagged" for destruction unless telomere length is regenerated. It is widely studied that the telomere synthesizing enzyme telomerase, quiescent in normal human somatic cells, is active in early embryonic and germ cells and undergoes reactivation in tumorigenic tissues upon regaining the hexameric sequence.^2,3 This sequence, whose diminution is responsible for the "end-replication problem" and subsequent targeting of cells for apoptosis, is of great interest in achieving therapeutic control of the tumorigenesis of certain cancerous cell lines. ^7,8

In order to effect control over telomerase function its genetic mechanism must be understood. The sequence has two core enzyme components required for activity: telomerase RNA (hTR) and telomerase reverse transcriptase (hTERT).⁷ Telomerase RNA provides the template for telomere repeat synthesis. Expression of hTERT is observed in malignant tumors and cancer cells but not in normal tissues or telomerase-negative cell lines, and a strong correlation is found between hTERT expression and telomerase activity in a variety of tumors. Such findings strongly suggest that the hTERT is a catalytic subunit homologue protein of human telomerase, and that upregulation of hTERT might be a critical event in carcinogenesis.¹ One oncogene that might activate TERT in the natural context is c-myc. Myc genes are frequently deregulated in human tumors and myc overexpression may cause telomerase reactivation and telomere stabilization, which in turn would allow permanent proliferation.⁷ Another protein potentially shown to regulate telomerase activity is protein phosphatase 2A (PP2A), which is responsible for dephosphorylating hTERT and inactivating telomerase. Abundant evidence indicates that regulation of telomerase is multi-factorial, occurring both transcriptionally and post-translationally.^{5, 9}

Specific interest has arisen in disruption of telomerase activity by sphingolipids. Ceramide, a sphingolipid bioeffector, is a molecule composed of sphingosine linked to a fatty acid via an amide linkage and is responsible for regulation of various agents. This hypothesis is supported by the presence of ceramide targets for direct action on the regulation of growth suppression; namely ceramide-activated protein phosphatase (CAPP). It has been shown experimentally that several stress agents regulate sphingolipid metabolism and cause ceramide to accumulate. Raising the intracellular ceramide concentration is sufficient to induce many of the stress responses associated with these agents, such as apoptosis and cell-cycle arrest. CAPP transduces the cellular actions of ceramide through several phosphatases by downstream inactivation of c-myc and activation of PP2A, which advocates ceramide as a cellular bioregulator of telomerase inhibition.^4,9

In a specific application of these principles deactivation of telomerase activity would be highly desirable in the disruption of tumorigenic lung cancers. Telomerase activity is detectable in 85% of lung cancers and its presence in tumors of patients with non-small cell lung carcinoma (NSCLC) is associated with a high cell proliferation rate and an advanced pathologic state. It has been shown that disrupting telomere stability via telomerase inhibition limits the proliferation of human cancer cells. Because lung cancer, having a less than 5% mean survival, is the leading cause of carcinomatous demise in the United States, the significant potential of telomerase suppression as a therapeutic target for this ailment is prodigious.⁹

Methods

TRAP assay: Telomerase activity has been detected in 85% of tumors of more than 20 various types of cancers tested by recent investigators. The TRAP assay is a highly sensitive, in vitro, one buffer, two enzyme system utilizing polymerase chain reaction (PCR) for large scale surveys of telomerase activity in human cells and tissues. In the first step of the reaction, telomerase adds a number of the telomeric repeats (GTTAGG) onto the 3’ end of a substrate oligonucleotide (TS). In the second step, the extended products are amplified by PCR using the TS and reverse primers (RP), generating a ladder of products with 6 base increments starting at 50 nucleotides: 50, 56, 62, 68, etc. Following amplification of the telomerase extract, the mix is run on a 10.0% or 12.5% polyacrylamide-gel electrophoresis (PAGE). The data is then analyzed by photographic or radiographic imaging.¹⁰

Cell lines and culture conditions: The A549 human lung adenocarcinoma cells are maintained in growth medium containing 10% fetal calf serum and 100ng/ml each of penicillin an streptomycin (Life Technologies; Grand Island, NY) at 37°C in 5% CO₂.⁹

Ceramides: Cell permeable and biologically active short chain ceramide, C6-ceramide, and its biologically inactive analog, dihydro-C6-ceramide, are obtained from the synthetic lipid core at the Department of Biochemistry and Molecular Biology, Medical University of South Carolina.⁹

Cell survival assay: The concentration of C6-ceramide that inhibited cell growth by 50% (IC₅₀) are determined from cell survival plots obtained by MTT cytotoxicity assay. The cells are plated into 96-well plates containing 100m l of the growth medium in the absence or presence of increasing concentrations of C6-ceramide at 37°C in 5% CO₂ for 24-48 hr. They are then treated with 25m l of 3-[4,5-dimethylthiazol-2-yl]-2-5-diphenyltetrazolium bromide (MTT) for 4-5 hr. After lysing the cells in 100m l of the lysis buffer, the plates are read in a micro plate reader (Dynatech, Chantilly, VA) at 570nm. After that, the IC₅₀ concentrations of C6-ceramide are determined from cell survival plots. Triplicate wells are used for each treatment. The final concentration of dimethyl sulfoxide (DMSO - a solvent for ceramide analogs) in the growth medium will be less than 0.1% (v/v) which has no effect on cell growth and survival.⁹

Trypan blue exclusion method: After being treated with C6-ceramide, the dead or dying cells are removed from the plates by phosphate buffered saline (PBS) washes. The remaining attached cells are trypsinized and then diluted in PBS. The dead and live cells are then counted using hematocytometer in the presence of trypan blue solution at 1:1 ratio (v/v) (Sigma Chemical Co.) as described by the manufacturer.⁹ The dead cells appear blue following uptake of the trypan solution, while the viable cells are portrayed as "clear;" a result of the dye-resistant integrity of the living plasma membranes.

Results

In order to examine the effect of ceramide on telomerase activity, A549 human lung adenocarcinoma cells were treated in the absence or presence of increasing concentrations of C6-ceramide for various time points. The dead cells that detached from the culture flasks were removed by PBS washes, and telomerase activity was measured in live cells (confirmed using trypan blue exclusion) by the PCR-based TRAP. First, serial dilutions of proteins (5-100ng) obtained from untreated A549 cell extracts were used in TRAP to establish a linear response range of the assay for reliable quantification of telomerase activity in these cells. Using ChemiImager, telomerase activity in each sample was quantitated by measuring total density of the bands present in the characteristic DNA ladder of the TRAP assay. The 32bp internal standard (IS) PCR product was used as a control for quantification. Treatment of the cell extracts with RNase or proteinase K eliminated telomerase activity, showing the specificity of the assay. No telomerase activity was detectable in samples containing 0-20ng of A549 cell extracts. Linear amplification was obtained using 50-100ng proteins in the TRAP assay. Therefore, 50-100ng of protein from each sample was used in subsequent TRAP assays.⁹

C6-ceramide inhibits telomerase: The treatment of A549 cells with 10-50m M C6-ceramide for 6-hr resulted in a dose dependent decrease in telomerase activity compared to untreated controls. The concentration of C6-ceramide that inhibited 50% telomerase activity was 20m M after 6-hr treatment. Moreover, C6-ceramide had no effect on telomerase activity in vitro, demonstrating that ceramide is indirectly involved in the inhibition of telomerase via mediating signaling pathways for regulation of its activity in vivo.⁹

Inhibition of telomerase by C6-ceramide is time dependent: To examine whether the inhibition of telomerase by C6-ceramide is time dependent, A549 cells were treated in the presence of 20m M C6-ceramide for 0, 3, 6 and 24 hr. The effect of C6-ceramide on the inhibition of telomerase activity was time dependent. Its activity was slightly decreased after 3-hr treatment with C6-ceramide. However, treatment of the cells with C6-ceramide for 6 and 24-hr resulted in a significant inhibition of telomerase activity (about 50% and 85%, respectively).⁹

Telomerase activity in response to C6-ceramide in the experiments above was measured using equal amounts of protein obtained from live cells that remained attached to the flasks, which was also confirmed by trypan blue exclusion. Moreover, to determine if the effective concentration of C6-ceramide (20m M) for the inhibition of telomerase activity in A549 cells is below its cytotoxic levels, MTT cytotoxicity assays were performed. A549 cells were grown with increasing concentrations of C6-ceramide for 48-hr, and then the IC₅₀ concentration of C6-ceramide that inhibited cell survival by 50% was determined from cell survival plots, and found to be 36m M. These results suggest that the inhibition of telomerase by 20m M C6-ceramide, which is below its toxic concentration, is not due to its general toxicity causing excessive cell death, but rather due to its inhibitory effect on telomerase.⁹

Treatment of A549 cells in the presence of 20m M C6-ceramide for 24-hr significantly inhibited telomerase activity compared to that of untreated controls, whereas treating cells with 20m M dihydro-C6-ceramide, a biologically inactive analog of C6-ceramide, for 24-hr had no effect on telomerase activity. These results show that the inhibition of telomerase activity by C6-ceramide is specific.⁹

Discussion

The presence of telomerase in nearly all cancers offers important therapeutic possibilities.⁸ The tumor suppressive action of ceramide in A549 human lung adenocarcinoma cells provides a potential pathway towards clinical control of the lethal lung cancers. The specificity of ceramide for inhibition of telomerase has been demonstrated by the time and dose dependent experimental methods described herein to measure linear telomerase activity. The use of dihydro-C6-ceramide confirmed specificity of biologically active ceramide by eliminating the possibility that telomerase suppression might have been precipitated by short-chain fatty acids in the prepared media.

Although the clinical possibilities of this study are encouraging, it is applicable to only one specific cell line. In order to verify the universality of the telomeric quelling properties of ceramide, cell lines from various tissues such as breast, pancreas and liver should be tested. Other possible targets for ceramide have been proposed including PKCx , PKCa , Raf and the GDP-GTP exchange factor Vav.⁴ The manipulation of telomerase could lead to tumorigenesis by imposing genetic instability, but the therapeutic potential for telomerase inhibitors provides a benefit worthy of the carcinogenic risk.³ Regardless, the development of therapeutic regimens founded on biochemical mechanisms will further the advancement of novel strategies for the treatment of human cancers.

References