Contributions
Abstract: PB1846
Type: Publication Only
Background
With advancing age, especially after the six decade of human life, the probability of developing cancer, including malignant hemopathies such as chronic lymphocytic leukemia (CLL), increases. This happens due to progressive decline in immunosurveillance that favors tumor escape and progression in elderly. With increasing age, the human body is more prone to genetic and epigenetic changes that are important contributing factors in the pathogenesis of cancer by negatively impacting on immune cell function. Moreover, these genetic or epigenetic modifications associated with aging could also impact on tumor suppressor genes (TSGs).
Aims
We previously reported a correlation between 6q deletion and progression into a T cell lymphoproliferative disease, identifying the BACH2 gene as a candidate TSG. We thus examined the expression of specific transcription factors; BACH2/PRDM1 in T and B cells for their potential role in immunosenescence.
Methods
Peripheral blood mononuclear cells were isolated from whole blood obtained from untreated B-CLL patients (n=41) and age-matched healthy donors (HD; n=60) using Lymphoprep (Stemcell Technologies) density gradient centrifugation. T cells (CD3+CD4+, CD3+CD8+) and B cells (CD19+) were purified (purity between 95%>99%) using magnetic isolation for subsequent molecular analyses. BACH2, PRDM1 and CDKN2A (p16INK4A) transcripts were quantified using RT-qPCR. BACH2 and BLIMP1 (PRDM1) protein expression were examined by Western blotting. To measure ß-galactosidase activity, we used flow cytometry with Fluorescein-di-beta-D-galactopyranoside (FDG) as a substrate for beta-galactosidase. T and B cell apoptosis was analyzed after intracellular oxidative stress-inducing etoposide treatment in both populations.
Results
BACH2 gene expression in the HD groups is significantly down-regulated in CD4+, CD8+ T cells and CD19+ B cells from the older HD group (p=0.0012; 0.0045 and 0.0367, respectively). BACH2 expression further reduced in CD4+, CD8+ T cells and CD19+ B cells from CLL patients compared to age matched-HD (p=0.001; <0.0001 and 0.0043). PRDM1 gene expression inversely correlated with BACH2 in CD4+, CD8+ T cells and CD19+ B cells (r=0.61; 0.71 and 0.65, respectively). Moreover, PRDM1 was as expected significantly upregulated in CD4+ and CD8+ T cells (p=0.0034; p=0.0017) from B-CLL patients but not in their leukemic-B cells. Western blotting analysis demonstrated that BACH2 and BLIMP1 (PRDM1) protein expressions in the T and B cell subpopulations significantly correlated with transcript expression. We further studied correlation between BACH2 expression and other senescence markers, such as p16INK4A (encoded by CDKN2A gene) and ß-galactosidase in both T cells and B cells. CDKN2A gene expression inversely correlated with BACH2 in CD4+, CD8+ T cells and CD19+ B cells (p=0.036; 0.025 and 0.043, respectively). ß-galactosidase activity showed a twofold increase compared to the BACH2 deficient lymphocytes (CD4+, CD8+ and CD19+) in older HD compared to young. We also observed a strong correlation between age-related BACH2 down-regulation and a decrease in CD4+ T cell and CD19+ B cell apoptosis (p=0.0127 and 0.0218 respectively).
Conclusion
Our data suggest that downregulation of BACH2 and upregulation of PRDM1 expression in major lymphocyte subsets from CLL patients and older healthy controls are significantly correlated with the aging immune cells and could be part of the resistance to apoptosis in B-CLL cells.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Chronic Lymphocytic Leukemia, Genetic instability, Senescence, transcription factor
Abstract: PB1846
Type: Publication Only
Background
With advancing age, especially after the six decade of human life, the probability of developing cancer, including malignant hemopathies such as chronic lymphocytic leukemia (CLL), increases. This happens due to progressive decline in immunosurveillance that favors tumor escape and progression in elderly. With increasing age, the human body is more prone to genetic and epigenetic changes that are important contributing factors in the pathogenesis of cancer by negatively impacting on immune cell function. Moreover, these genetic or epigenetic modifications associated with aging could also impact on tumor suppressor genes (TSGs).
Aims
We previously reported a correlation between 6q deletion and progression into a T cell lymphoproliferative disease, identifying the BACH2 gene as a candidate TSG. We thus examined the expression of specific transcription factors; BACH2/PRDM1 in T and B cells for their potential role in immunosenescence.
Methods
Peripheral blood mononuclear cells were isolated from whole blood obtained from untreated B-CLL patients (n=41) and age-matched healthy donors (HD; n=60) using Lymphoprep (Stemcell Technologies) density gradient centrifugation. T cells (CD3+CD4+, CD3+CD8+) and B cells (CD19+) were purified (purity between 95%>99%) using magnetic isolation for subsequent molecular analyses. BACH2, PRDM1 and CDKN2A (p16INK4A) transcripts were quantified using RT-qPCR. BACH2 and BLIMP1 (PRDM1) protein expression were examined by Western blotting. To measure ß-galactosidase activity, we used flow cytometry with Fluorescein-di-beta-D-galactopyranoside (FDG) as a substrate for beta-galactosidase. T and B cell apoptosis was analyzed after intracellular oxidative stress-inducing etoposide treatment in both populations.
Results
BACH2 gene expression in the HD groups is significantly down-regulated in CD4+, CD8+ T cells and CD19+ B cells from the older HD group (p=0.0012; 0.0045 and 0.0367, respectively). BACH2 expression further reduced in CD4+, CD8+ T cells and CD19+ B cells from CLL patients compared to age matched-HD (p=0.001; <0.0001 and 0.0043). PRDM1 gene expression inversely correlated with BACH2 in CD4+, CD8+ T cells and CD19+ B cells (r=0.61; 0.71 and 0.65, respectively). Moreover, PRDM1 was as expected significantly upregulated in CD4+ and CD8+ T cells (p=0.0034; p=0.0017) from B-CLL patients but not in their leukemic-B cells. Western blotting analysis demonstrated that BACH2 and BLIMP1 (PRDM1) protein expressions in the T and B cell subpopulations significantly correlated with transcript expression. We further studied correlation between BACH2 expression and other senescence markers, such as p16INK4A (encoded by CDKN2A gene) and ß-galactosidase in both T cells and B cells. CDKN2A gene expression inversely correlated with BACH2 in CD4+, CD8+ T cells and CD19+ B cells (p=0.036; 0.025 and 0.043, respectively). ß-galactosidase activity showed a twofold increase compared to the BACH2 deficient lymphocytes (CD4+, CD8+ and CD19+) in older HD compared to young. We also observed a strong correlation between age-related BACH2 down-regulation and a decrease in CD4+ T cell and CD19+ B cell apoptosis (p=0.0127 and 0.0218 respectively).
Conclusion
Our data suggest that downregulation of BACH2 and upregulation of PRDM1 expression in major lymphocyte subsets from CLL patients and older healthy controls are significantly correlated with the aging immune cells and could be part of the resistance to apoptosis in B-CLL cells.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Chronic Lymphocytic Leukemia, Genetic instability, Senescence, transcription factor