Accelerated replicative senescence of the peripheral immune system induced by HIV infection.

OBJECTIVES: HIV induces rapid turnover of T lymphocytes but whether this leads to replicative senescence of CD4+ and CD8+ cells and contributes to AIDS symptoms is unclear. The aim of this study was to address this question by analyzing telomere length in blood cell populations as a measure of replicative history in a significant number of patients infected with HIV. DESIGN: Total peripheral blood mononuclear cells (PBMCs), CD4+ or CD8+ cells were isolated from blood collected from a total of 73 HIV patients and 27 controls. Samples were isolated to measure telomere length, telomerase activity and proliferative ability, and analyses were carried out in a blind experimental protocol. METHODS: PBMCs isolated on Ficoll-Hypaque gradients were washed and prepared for additional fractionation into CD4+ and CD8+ cells using antibody-bound magnetic beads. Total PBMCs, CD4+ and CD8+ cells were used for cell cycle analysis, for telomerase activity assays and were measured for telomere length using the terminal restriction fragment assay. RESULTS: Telomere analyses in this study show a clear (P < 0.0001) inverse relationship between telomere length and progression of immunosuppression, with HIV infection resulting in a five-fold or greater acceleration of aging of the circulating PBMC component of the immune system. Patients who are 37 years old showed telomere lengths similar to uninfected 75-year-olds. Telomere loss correlated well with progression of AIDS and with reduced proliferative ability of patient PBMCs but was unrelated to telomerase activity. Mean telomere length was shorter in both CD4+ and CD8+ cells, with three-fold higher rates of telomere loss for CD8+ lymphocytes. CONCLUSIONS: These data provide strong support for the occurrence of accelerated replicative aging of the peripheral immune system, possibly resulting in a loss of T cells leading to AIDS symptoms.

A role for serine proteases in mediating phorbol ester-induced differentiation of HL-60 cells.

Treatment of human HL-60 promyelocytic leukemia cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) results in increases in proteolytic activity and maturation toward the monocytic lineage. To investigate the potential roles that different classes of proteases play in the monocytic differentiation of HL-60 cells, cells were treated with phorbol ester in the presence of various serine and cysteine protease inhibitors. The serine protease inhibitors 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), N-alpha-tosyl-phenylalanine chloromethyl ketone (TPCK), and N-alpha-tosyl-lysine chloromethyl ketone (TLCK) repressed a number of phenotypic markers of monocytic differentiation including surface expression of the CD11b integrin, cell aggregate formation, cell cycle exit, and cell death. CD11b was not detected at the cell surface by FACS analysis up to 24 h after induction of differentiation; however, both CD11b mRNA and protein were present. Downregulation of c-myc mRNA and upregulation of c-fos and egr-1 mRNA and protein, which normally occur during TPA-induced differentiation, were not affected by inclusion of the protease inhibitors. These data indicate that serine proteases specifically mediate many of the phenotypic aspects of TPA-induced monocytic differentiation but are not involved with the induction or repression of differentiation-sensitive transcription factors and suggest that serine protease activity is required for intracellular processing of CD11b.

Selective inhibition of telomerase activity during terminal differentiation of immortal cell lines.

Telomerase, the enzyme that maintains the ends of linear eukaryotic chromosomes, is active in human germ cells and in a majority of tumor tissues and immortalized cell lines. In contrast, most mature somatic cells and tissues contain low or undetectable telomerase activity, implying a stringent negative regulatory control mechanism. We report here that telomerase activity is dramatically inhibited during the terminal differentiation of HL-60 human promyelocytic leukemia cells to monocytic and granulocytic lineages. A loss of telomerase activity was seen in response to three different inducers of differentiation, was independent of differentiation-induced apoptosis, and occurred in the presence of unaltered expression of the RNA component of telomerase. Reduction in telomerase activity was also observed during the differentiation of murine F9 teratocarcinoma and C2C12 myoblast cells. In contrast, induced differentiation of murine p19 embryonal carcinoma and Neuro 2a neuroblastoma cells did not result in a loss of telomerase activity. These results are therefore consistent with the absence of telomerase activity in human somatic cells and the presence of telomerase activity in many somatic murine cells and tissues.