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Differential PAX5 levels promote malignant B-cell infiltration, progression and drug resistance, and predict a poor prognosis in MCL patients independent of CCND1.

Teo AE, Chen Z, Miranda RN, McDonnell T, Medeiros LJ, McCarty N - Leukemia (2015)

Bottom Line: On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6.Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells.Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation, Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), University of Texas-Health Science Center at Houston, Houston, TX, USA.

ABSTRACT
Reduced Paired box 5 (PAX5) levels have important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5(-) MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to the bone marrow stromal cells and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb, and to phosphoinositide 3-kinase/mammalian target of rapamycin and phosphorylated signal transducer and activator of transcription 3 pathway hyperactivation. On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

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PAX5 silencing in MCL cells leads to increased cell proliferation, and PAX5 overexpression leads to cell death(A) PAX5− cells were initially seeded at 2×103 cells and were cultured for 7 days. Viable cells were counted using Trypan Blue staining at days 0, 2, 4 and 7. Each value represents the mean ± S.D. (n=3). (B) PAX5− cells (5×104) were cultured under two different low-serum conditions and were cultured for 7 days. Viable cells were counted using Trypan Blue staining at days 0, 2, 4 and 7. Each value represents the mean ± S.D. (n=3). (C) qPCR analysis of cell cycle-related genes in PAX5− cells. The values were normalized to β-actin. Each qRT value represents the mean ± S.D. (n=3). (D) Semiquantitative RT-PCR analysis showing the dysregulation of potential cell cycle inhibitors upon PAX5 silencing. SP53 cells harbor wild type TP53, and Jeko cells harbor a TP53 deletion mutant. GAPDH served as a loading control. (E) Immunoblot analyses of the cell cycle inhibitors p53, p27 and p21 in PAX5− and control cells; β-actin served as the loading control. All cells used in these experiments were selected based on positive GFP expression, and stable cell lines were generated via antibiotic selection. *p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).
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Figure 1: PAX5 silencing in MCL cells leads to increased cell proliferation, and PAX5 overexpression leads to cell death(A) PAX5− cells were initially seeded at 2×103 cells and were cultured for 7 days. Viable cells were counted using Trypan Blue staining at days 0, 2, 4 and 7. Each value represents the mean ± S.D. (n=3). (B) PAX5− cells (5×104) were cultured under two different low-serum conditions and were cultured for 7 days. Viable cells were counted using Trypan Blue staining at days 0, 2, 4 and 7. Each value represents the mean ± S.D. (n=3). (C) qPCR analysis of cell cycle-related genes in PAX5− cells. The values were normalized to β-actin. Each qRT value represents the mean ± S.D. (n=3). (D) Semiquantitative RT-PCR analysis showing the dysregulation of potential cell cycle inhibitors upon PAX5 silencing. SP53 cells harbor wild type TP53, and Jeko cells harbor a TP53 deletion mutant. GAPDH served as a loading control. (E) Immunoblot analyses of the cell cycle inhibitors p53, p27 and p21 in PAX5− and control cells; β-actin served as the loading control. All cells used in these experiments were selected based on positive GFP expression, and stable cell lines were generated via antibiotic selection. *p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).

Mentions: We generated two cell lines in which PAX5 was stably knocked down: PAX5− Jeko and PAX5− SP53 MCL cells. Lentiviral PAX5 shRNA transduction efficiently silenced PAX5 expression in GFP+ SP53 and Jeko cells (Supplementary Figure 1a), whereas PAX5 ORF lentiviral infection generated PAX5-overexpressing cells (Supplementary Figure 1b). We next compared the proliferation of PAX5− MCL cells to that of control cells transfected with scrambled RNA. Unexpectedly, cell proliferation was significantly increased in PAX5− MCL cells (Figure 1a). In contrast, PAX5 overexpression (PAX5ORF) severely reduced cell proliferation (Figure 1b) and increased apoptosis in these cells (Supplementary Figure 1c). PAX5− MCL cells also thrived under serum-starved conditions; PAX5− MCL cells exhibited significantly increased cell survival in 2% or 5% serum (Figure 1b; Supplementary Figure 1d), indicating that silencing PAX5 promotes MCL cell survival not only under normal cell culture conditions but also under stressed conditions.


Differential PAX5 levels promote malignant B-cell infiltration, progression and drug resistance, and predict a poor prognosis in MCL patients independent of CCND1.

Teo AE, Chen Z, Miranda RN, McDonnell T, Medeiros LJ, McCarty N - Leukemia (2015)

PAX5 silencing in MCL cells leads to increased cell proliferation, and PAX5 overexpression leads to cell death(A) PAX5− cells were initially seeded at 2×103 cells and were cultured for 7 days. Viable cells were counted using Trypan Blue staining at days 0, 2, 4 and 7. Each value represents the mean ± S.D. (n=3). (B) PAX5− cells (5×104) were cultured under two different low-serum conditions and were cultured for 7 days. Viable cells were counted using Trypan Blue staining at days 0, 2, 4 and 7. Each value represents the mean ± S.D. (n=3). (C) qPCR analysis of cell cycle-related genes in PAX5− cells. The values were normalized to β-actin. Each qRT value represents the mean ± S.D. (n=3). (D) Semiquantitative RT-PCR analysis showing the dysregulation of potential cell cycle inhibitors upon PAX5 silencing. SP53 cells harbor wild type TP53, and Jeko cells harbor a TP53 deletion mutant. GAPDH served as a loading control. (E) Immunoblot analyses of the cell cycle inhibitors p53, p27 and p21 in PAX5− and control cells; β-actin served as the loading control. All cells used in these experiments were selected based on positive GFP expression, and stable cell lines were generated via antibiotic selection. *p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).
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Figure 1: PAX5 silencing in MCL cells leads to increased cell proliferation, and PAX5 overexpression leads to cell death(A) PAX5− cells were initially seeded at 2×103 cells and were cultured for 7 days. Viable cells were counted using Trypan Blue staining at days 0, 2, 4 and 7. Each value represents the mean ± S.D. (n=3). (B) PAX5− cells (5×104) were cultured under two different low-serum conditions and were cultured for 7 days. Viable cells were counted using Trypan Blue staining at days 0, 2, 4 and 7. Each value represents the mean ± S.D. (n=3). (C) qPCR analysis of cell cycle-related genes in PAX5− cells. The values were normalized to β-actin. Each qRT value represents the mean ± S.D. (n=3). (D) Semiquantitative RT-PCR analysis showing the dysregulation of potential cell cycle inhibitors upon PAX5 silencing. SP53 cells harbor wild type TP53, and Jeko cells harbor a TP53 deletion mutant. GAPDH served as a loading control. (E) Immunoblot analyses of the cell cycle inhibitors p53, p27 and p21 in PAX5− and control cells; β-actin served as the loading control. All cells used in these experiments were selected based on positive GFP expression, and stable cell lines were generated via antibiotic selection. *p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).
Mentions: We generated two cell lines in which PAX5 was stably knocked down: PAX5− Jeko and PAX5− SP53 MCL cells. Lentiviral PAX5 shRNA transduction efficiently silenced PAX5 expression in GFP+ SP53 and Jeko cells (Supplementary Figure 1a), whereas PAX5 ORF lentiviral infection generated PAX5-overexpressing cells (Supplementary Figure 1b). We next compared the proliferation of PAX5− MCL cells to that of control cells transfected with scrambled RNA. Unexpectedly, cell proliferation was significantly increased in PAX5− MCL cells (Figure 1a). In contrast, PAX5 overexpression (PAX5ORF) severely reduced cell proliferation (Figure 1b) and increased apoptosis in these cells (Supplementary Figure 1c). PAX5− MCL cells also thrived under serum-starved conditions; PAX5− MCL cells exhibited significantly increased cell survival in 2% or 5% serum (Figure 1b; Supplementary Figure 1d), indicating that silencing PAX5 promotes MCL cell survival not only under normal cell culture conditions but also under stressed conditions.

Bottom Line: On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6.Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells.Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation, Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), University of Texas-Health Science Center at Houston, Houston, TX, USA.

ABSTRACT
Reduced Paired box 5 (PAX5) levels have important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5(-) MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to the bone marrow stromal cells and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb, and to phosphoinositide 3-kinase/mammalian target of rapamycin and phosphorylated signal transducer and activator of transcription 3 pathway hyperactivation. On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

Show MeSH
Related in: MedlinePlus