Early and Permanent Telomere Shortening in Bone Marrow-Derived Cells Following Chemotherapy: A Parallel Study In Vivo in Lymphoma Patients and In Vitro in Cultured Mesenchymal Stem Cells

Patients with lymphoproliferative disease very often require treatments with chemotherapy. Several recent reports suggest that the exposure of hematopoietic cells to DNA-damaging substances, such as chemotherapeutic drugs, may trigger the ageing process and induce premature cell ageing. A good indicator of cell replication history and thus of cell ageing is the length of telomeres. Indeed, telomere shortening and/or telomere dysfunction have been documented in patients receiving chemotherapy. The loss of telomere sequences has been linked to the increased risk of developing secondary malignancy in subjects previously treated with chemotherapy. However, it is still unexplained whether telomere loss is secondary to direct damage of bone marrow (BM) cells by cytotoxic drugs, or merely the consequence of increased BM cell proliferation after exposure to chemotherapy. Moreover, the time course for the onset of chemotherapy-induced telomere shortening and the cell types mainly involved in the premature cell aging induced by chemotherapy remain to be elucidated. In the present study changes in telomere length (TL) following chemotherapy were evaluated both in vivo and in vitro. Main aims of the study were: i. to define whether TL shortening following chemotherapy is reversible or permanent; ii. to verify whether TL shortening is a phenomenon induced by extensive chemotherapy treatments or it may occur even after minimal drug exposures; iii. to identify possible cell populations that are particularly susceptible to drug-induced telomere loss. Patients and methods Mononuclear Cells (MNC) or granulocytes were obtained from BM and/or Peripheral blood (PB) cells from 31 lymphoma patients treated with high-dose chemotherapy and autograft and from 14 lymphoma patients undergoing chemotherapy (6 CHOP, 1 CVP, 1 MINE, 1 DHAP and 5 ABVD). Overall, 27 patients of the autograft and 13 patients of the conventional chemotherapy groups were at their first treatment line. Median age of patients was 45 years. TL was assessed on granulocytes and MNC by Southern Blotting, as previously reported (Ricca et al, Leukemia 2005). In vitro studies were performed on cultured mesenchymal stem cells (MSCs). Briefly, MNC were obtained from BM of normal volunteers or patients undergoing routine diagnostic procedures; cells were seeded in MEM-alpha medium and 10% platelet lysates and fed at 3–4 day intervals; cultured MSC were identified for positivity of CD105, CD90, CD29, CD44 and then assayed for response to DNA-damaging drugs between the second and the third passage in culture. Two chemotherapeutic drugs were employed, Doxorubicin (Doxo) and etoposide (Eto). Cells were incubated for 2 hours with decreasing doses of the tested drugs, and 10 nM Doxo and 500 ng/ml Eto were the highest doses of the drugs that were used without any distress on cell proliferation and cell viability. The 2-hour exposure to chemotherapy was repeated at 7 day intervals up to four times. TL was evaluated both by flow-fish and southern-blot analysis. Results A marked reduction in TL was detected in all patients undergoing autograft in PB granulocytes and in BM cells, compared to age-matched controls; the degree of TL loss remained detectable even in patients up to 10 yrs. since autograft. For patients receiving conventional chemotherapy, a significant TL shortening could be detected in granulocyte obtained after chemotherapy compared to pre-treatment values (p=0.029), while no significant variations could be documented in MNC. TL shortening was detectable already after the first chemotherapy course in six patients and at the second in four patients. A marked TL shortening occurred in cultured MSC after exposure to sub-lethal doses of Doxo and Eto. Initial TL shortening was detectable already at 5 days after drug exposure, with progressive reduction compared with untreated cells at 7, 14, 21, and 28 days in culture. Following a single exposure, MSCs were unable to regain the lost telomere sequences for up to 28 days in culture. Conclusions The results indicate that TL shortening: i. is a permanent signature of the previous DNA damage in BM cells exposed to chemotherapy; ii. is a phenomenon that can be detected early following chemotherapy exposure, even with low drug dosages; iii. can be most easily detectable in myeloid cells, in particular in granulocytes, although also BM mesenchymal cells may be susceptible to drug-induced telomere loss.

No relevant conflicts of interest to declare.