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Different effects of interferon-alpha on melanoma cell lines: a study on telomerase reverse transcriptase, telomerase
E Maellaro1, L Pacenti, B Del Bello
1Department of Pathophysiology and Experimental Medicine, University of Siena, Italy.
Background:
Although the antiproliferative and proapoptotic effects of interferon (IFN)-alpha are widely recognized, its antitumour mechanisms are not completely known. Recent studies indicate that the derepressed expression of the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), and telomerase activity (TA) are involved in the process of human carcinogenesis. Only a few studies have investigated the effects of IFN-alpha on hTERT and TA, with controversial results. Objectives To study the hTERT mRNA expression, TA and apoptosis in human melanoma cells treated with IFN-alpha.
Methods:
Five human melanoma cell lines (Me665/2/21, Me665/2/60, HT-144, SK-Mel-28 and SK-Mel-5) were cultured in standard conditions and treated with 20000 IU mL-1 of human recombinant IFN-alpha-2b. Apoptosis was evaluated as hypodiploid DNA content determined by flow cytometry, caspase-3/7 activity by enzymatic assay, and poly(adenosine diphosphate-ribose) polymerase cleavage by Western blot analysis. IFN-alpha receptor (IFNA-R) and hTERT mRNA expression levels were evaluated by semiquantitative reverse transcription-polymerase chain reaction. TA was evaluated by a polymerase chain reaction-based telomerase repeat amplification protocol assay.
Results:
Besides a variable degree of cell proliferation inhibition in all cell lines tested, we found different responses, ranging from no significant effects in SK-Mel-28 cells, to a high degree of apoptosis with no hTERT mRNA expression and TA modification in HT-144 cells, and induction of apoptosis, along with decrease in hTERT mRNA expression and TA in Me665/2/21 cells. No induction of apoptosis was observed in SK-Mel-5 and Me665/2/60 cells, although an early decrease in hTERT mRNA expression, and a minor increase of both hTERT mRNA expression and TA were found, respectively.
Conclusions:
Our results suggest that the effects of IFN-alpha on hTERT and TA can result from the induction of apoptosis, but they can also occur through a direct modulation of hTERT. We hypothesize that, depending on the cellular context rather than the IFNA-R status of the targeted cells, IFN-alpha can elicit an apoptotic cell death; furthermore, different pathways of apoptosis, not necessarily involving telomerase, can be put into motion.
Insights
Interferon-alpha (IFN-alpha) affects human melanoma cells by inducing apoptosis and modulating telomerase activity (TA). These effects on human telomerase reverse transcriptase (hTERT) expression and TA depend on the cellular context, not just the IFN-alpha receptor status.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Interferon-alpha (IFN-alpha) exhibits antiproliferative and proapoptotic effects, but its precise anti-cancer mechanisms remain unclear.
- Deregulation of human telomerase reverse transcriptase (hTERT) and telomerase activity (TA) are implicated in human carcinogenesis.
- Previous studies on IFN-alpha's impact on hTERT and TA have yielded conflicting results.
Purpose of the Study:
- To investigate the effects of IFN-alpha on hTERT mRNA expression, TA, and apoptosis in human melanoma cell lines.
- To explore the relationship between IFN-alpha treatment, apoptosis induction, and telomerase modulation in melanoma.
- To understand the differential responses of various melanoma cell lines to IFN-alpha.
Main Methods:
- Cultured five human melanoma cell lines and treated them with human recombinant IFN-alpha-2b.
- Assessed apoptosis via hypodiploid DNA content, caspase-3/7 activity, and poly(ADP-ribose) polymerase cleavage.
- Quantified IFN-alpha receptor (IFNA-R) and hTERT mRNA expression using RT-PCR, and TA using a PCR-based assay.
Main Results:
- Observed variable inhibition of cell proliferation across all tested cell lines.
- HT-144 cells showed high apoptosis with no change in hTERT mRNA or TA.
- Me665/2/21 cells exhibited apoptosis with decreased hTERT mRNA and TA; SK-Mel-5 and Me665/2/60 cells showed no apoptosis but early decreases or minor increases in hTERT mRNA and TA, respectively.
Conclusions:
- IFN-alpha's effects on hTERT and TA can be mediated by apoptosis induction or direct hTERT modulation.
- The cellular context, rather than IFNA-R status, dictates IFN-alpha's ability to induce apoptosis.
- Melanoma cells may undergo apoptosis via pathways independent of telomerase modulation in response to IFN-alpha.