Telomerase activity and telomeric length remain unchanged in JurkaT-cells infected with HIV-1

MINCES LR; REYNOSO RP; BOLCIC FM; SALOMON H; QUARLERI JF

Rio de Janeiro, Brasil

Congreso; 3rd IAS Conference on HIV Pathogenesis and Treatment.; 2005

International AIDS Society

Introduction: The pathogenesis of HIV-1 infection at a molecular level has not been completely elucidated yet. We aim to evaluate the in vitro impact of HIV-1 infection on cellular senescence.The pathogenesis of HIV-1 infection at a molecular level has not been completely elucidated yet. We aim to evaluate the in vitro impact of HIV-1 infection on cellular senescence. Methods: An HIV-1 BH10 strain was used to infect E6-JurkaT-cells (T Leukemia derivate line with high intrinsic telomeric activity). Ninety-six hours later cells were harvested and split for telomerase activity quantification (Roche Diagnostics, Germany), flow cytometry evaluation of apoptosis-necrosis degree by Annexin V and 7-AAD, and telomeric length analysis (TRF-telomeric restriction fragment). Culture supernatants were collected to quantify HIV-1 p24 antigen (Coulter Corporation, USA) as a measurement of productive infection. All experiments were carried out by duplicate and also included uninfected cells in each assay as a negative control. Statistic analysis was done using a non parametric test.An HIV-1 BH10 strain was used to infect E6-JurkaT-cells (T Leukemia derivate line with high intrinsic telomeric activity). Ninety-six hours later cells were harvested and split for telomerase activity quantification (Roche Diagnostics, Germany), flow cytometry evaluation of apoptosis-necrosis degree by Annexin V and 7-AAD, and telomeric length analysis (TRF-telomeric restriction fragment). Culture supernatants were collected to quantify HIV-1 p24 antigen (Coulter Corporation, USA) as a measurement of productive infection. All experiments were carried out by duplicate and also included uninfected cells in each assay as a negative control. Statistic analysis was done using a non parametric test. Results: After 96 hr p.i., the average amount of p24 antigen in supernatant was 230 ng/ml in the infected cultures and not detectable in the uninfected control. The percentage of viable cells [Annexin V (-) and 7-AAD (-)] was 90%. The average telomerase activity (expressed as Relative Telomerase Activity) showed not significant variation in HIV-1 infected JurkaT-cells when compared to non-infected cells (p<0.05). Telomeric length was not reduced in infected cells when compared to the control.After 96 hr p.i., the average amount of p24 antigen in supernatant was 230 ng/ml in the infected cultures and not detectable in the uninfected control. The percentage of viable cells [Annexin V (-) and 7-AAD (-)] was 90%. The average telomerase activity (expressed as Relative Telomerase Activity) showed not significant variation in HIV-1 infected JurkaT-cells when compared to non-infected cells (p<0.05). Telomeric length was not reduced in infected cells when compared to the control. Conclusions: Considering that neither telomerase activity was reduced nor telomeric length was shortened in infected leukemic cells, we can infer that HIV-1 does not have a measurable impact on senescence in our in vitro model. Since these results are not congruent with previously reported findings in other cellular models, we hypothesize that, although HIV-1 infection might down-regulate telomerase activity in JurkaT-cells, it is not reflected by using the present experimental conditions due to the elevated intrinsic telomerase activity that this cell line displays.Considering that neither telomerase activity was reduced nor telomeric length was shortened in infected leukemic cells, we can infer that HIV-1 does not have a measurable impact on senescence in our in vitro model. Since these results are not congruent with previously reported findings in other cellular models, we hypothesize that, although HIV-1 infection might down-regulate telomerase activity in JurkaT-cells, it is not reflected by using the present experimental conditions due to the elevated intrinsic telomerase activity that this cell line displays.