Low telomerase activity: Possible role in the progression of human medullary thyroid carcinoma

Abstract

Maintenance of telomere length has been reported to be an absolute requirement for unlimited growth of human tumour cells and in about 85% of cases, this is achieved by reactivation of telomerase, the enzyme that elongates telomeres. Only in rare cases, like in human medullary thyroid carcinomas (MTC), telomerase activity (TA) is low or undetectable; however, this does not limit tumours to become clinically significant. Here, we report that very low TA (below 5% of HEK293) observed in MTC cell strains derived from different patients, although not sufficient for immortalising the cells, is necessary for prolonging their replicative life span. Telomere erosion led to induction of a crisis period after long-term in vitro cultivation, which was reached earlier when treating the cells with MST-312, a telomerase inhibitor at non-toxic concentrations. Crisis was bypassed either by ectopic hTERT introduction or by infrequent spontaneous immortalisation, the latter of which was always associated with telomerase reactivation and changes of the cellular phenotype. While confirming the high importance of telomerase for tumour development, these data draw attention to the relevance of low TA: although insufficient for telomere stabilisation, it allows MTC cells to reach more population doublings, increasing both cell numbers as well as the risk of accumulating mutations and thus might support the development of clinically significant MTC.

Introduction

Normal human cells can only be propagated for a limited number of population doublings (PDs) in vitro before entering the phase of replicative senescence¹ This senescent state is supposed to be the result of progressive telomere shortening with each cell division,² because critically short telomeres are recognised as DNA damage and subsequently induce this irreversible growth arrest.³ In contrast, most human tumour cells have gained a mechanism to prevent telomere erosion, which is usually achieved by reactivation of telomerase, a ribonucleoprotein enzyme complex that elongates telomeres.⁴ Since approximately 85% of tumours are characterised by elevated telomerase activity (TA), TA can be regarded as the most universal characteristic marker for human cancers.⁵ However, besides telomerase reactivation, an alternative telomere lengthening mechanism (ALT) has been described as well,⁶ which is discussed to be based on homologous recombination.⁷ Recent data also demonstrate that telomere maintenance in human cells may be more diverse than previously thought.8, 9, 10, 11, 12 As described recently, human liposarcomas, although showing an incidence for ALT and to the same extent for telomerase reactivation, can also develop without, or with a yet unknown, telomere maintenance mechanism.¹³ Similar results have been shown for a non-small cell lung cancer cell line⁸ as well as thyroid neoplasia.¹⁴ This raises the question as to whether a constitutive telomere length maintenance mechanism is a prerequisite for all tumour cells to form clinically significant tumours.

In order to address this question, we have used previously established, well characterised human medullary thyroid carcinoma (MTC) cell strains derived from eight different patients.15, 16, 17 MTC is a rare tumour arising from parafollicular C cells of the thyroid gland which in many cases is characterised by low to undetectable TA in vivo.18, 19, 20, 21, 22, 23, 24, 25, 26 Accordingly, seven out of eight cell strains showed low TA at early population doubling level (PDL) after establishment of primary cell cultures.

Here we present evidence that even this low TA is sufficient to increase the replicative life span of human MTC cells and therefore its inhibition might contribute to reducing the size of tumours in vivo.