Telomerase activity of human peripheral blood mononuclear cells in the course of HTLV type 1 infection in vitro.

Abstract

249 TELOMERASE IS INVOLVED in cell immortalization, and is expressed in stem cells of regenerating tissues, in lymphocytes subjected to mitogenic stimuli, and in cancer cells.1–3 The enzymatic complex (i.e., the RNA template [hTR] component and the reverse transcriptase [hTERT] catalytic subunit) restores telomere loss occurring at each cell division.4 The acquisition of “replicative immortality” in differentiated cells is often a critical step in malignant transformation. In this context, the appearance of telomerase activity can be considered one of the major biochemical steps that are required, although not sufficient per se, for oncogenic transformation. Human T cell leukemia virus type 1 (HTLV-1) is a human lymphotropic retrovirus associated with chronic adult T cell leukemia (ATL) widely diffused in endemic areas of Japan and South America.5 The outcome of the infection kinetics and selection of the immortalized clone that leads to the development of ATL can be experimentally monitored in vitro. In fact, HTLV-1 can preferentially transform CD4 T cells of peripheral blood mononuclear cells (PBMCs) obtained from healthy donors.6 A key step in HTLV-1 induced leukemogenesis is the induction of abnormal T cell growth, and immortalization of target lymphocytes. Therefore, it is conceivable that telomerase function could be activated in infected T cells. Actually, it has been demonstrated that a correlation exists between telomerase activity and progression of ATL.7 In addition, HTLV-2 was found to induce telomerase activity in short-term cultures of hematopoietic precursors,8 although the experimental conditions adopted in this study are entirely different from those utilized in the present investigation. All these observations prompted us to investigate how telomerase is activated in PBMCs during HTLV-1 infection in vitro, in order to obtain preliminary information about the complex interrelationship between viral leukemogenesis and host cell immortalization. Cells exposed to the virus generally proliferate during the first 3–4 weeks after infection. They then undergo a “growth crisis,” entering a quiescent phase for several weeks. Subsequently, surviving cells reenter a growth phase and become immortalized.6 Therefore it is reasonable to hypothesize that the immortalization step in HTLV-1-infected clones could be characterized by telomerase reactivation. To evaluate whether telomerase activity would be associated with the clonal selection of potentially immortalized cell clones, this enzymatic activity was studied in PBMC/MT-2 cocultures at various times postinfection. The results of this study, in line with previous observations, show that resting PBMCs cultivated in the presence of interleukin 2 (IL-2), but not exposed to the virus, survived in vitro for a few weeks. In contrast, after exposure to HTLV-1, PBMCs underwent a proliferative response, presumably due to the virus itself, and to the allogeneic stimulation prompted by the HTLV1 donor MT-2 cells (see the results of a representative experiment, confirmed at least three times, in Fig. 1). Thereafter, proliferation declined and, as expected, a growth crisis occurred in cocultured PBMCs (Fig. 1A). At approximately 8–9 weeks postinfection, a “clonal expansion phase” is frequently but not always observed. Actually, in a certain number of cases, PBMCs seem to control HTLV-1 infection with high efficiency, and the emergence of immortalized clones does not occur under the present standard culture conditions. However, in this study, in all three cases, immortalized infected cell lines were successfully obtained. In the representative experiment illustrated in Fig. 1, cells exposed to HTLV-1 overcame growth crisis starting from week 9 of culture (Fig. 1A). Progressive viral infection of the coculture, monitored by the expression of HTLV-1 Tax protein (Western blot) was also observed 2, 4, and 6 weeks postinfection, reaching a steady state by 13 weeks postinfection (Fig. 1B). It must be pointed out that until week 4 of the coculture the ratio of CD4versus CD8-positive cells was about 2:1. Ten weeks postinfection this ratio was about 10:1 (data not shown). Telomerase activity was tested by telomeric repeat amplification protocol (TRAP) assay,9 using equal numbers of viable