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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− cells demonstrate increased colony formation and PKH26+ cell retention in xenografted mice(A) PAX5− cells (5×103) were seeded in PHA-LCM, and colonies were scored at day 7. Only colonies containing > 50 cells were assigned a positive score. Inset: PAX5− cells also formed larger colonies in PHA-LCM, as visualized using a light microscope with a 20x objective. Each value represents the mean number of counted colonies ± S.D. (n=3). Lower panel: The counted colonies of SP53 cells (Left) and Jeko cells (Right) were represented in a graph. PAX5− cells exhibited higher mean numbers of colonies formed ± S.D. (n=3). (B) PAX5− cells displayed increased PKH26+ cell retention in xenografted mice. PKH26+ GFP+ PAX5− cells (1×106) were I.V. transferred to NOD/SCID mice. The mice were sacrificed 48 hours after injection, and cells were collected from bone marrow (femurs and tibias) and the spleen. The FACS analysis results are representative of 4 biological replicates. (C) Representative confocal images of GFP- and PKH-positive cells are shown. The images are merged from three different channels (DAPI, RFP and GFP) to better visualize and contrast human cells in a murine microenvironment. The arrows indicate GFP- and human PKH (RFP)-positive cells, and all cells were stained with the nucleic acid stain DAPI. Scale bar, 75 µm.*p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).
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Figure 2: PAX5− cells demonstrate increased colony formation and PKH26+ cell retention in xenografted mice(A) PAX5− cells (5×103) were seeded in PHA-LCM, and colonies were scored at day 7. Only colonies containing > 50 cells were assigned a positive score. Inset: PAX5− cells also formed larger colonies in PHA-LCM, as visualized using a light microscope with a 20x objective. Each value represents the mean number of counted colonies ± S.D. (n=3). Lower panel: The counted colonies of SP53 cells (Left) and Jeko cells (Right) were represented in a graph. PAX5− cells exhibited higher mean numbers of colonies formed ± S.D. (n=3). (B) PAX5− cells displayed increased PKH26+ cell retention in xenografted mice. PKH26+ GFP+ PAX5− cells (1×106) were I.V. transferred to NOD/SCID mice. The mice were sacrificed 48 hours after injection, and cells were collected from bone marrow (femurs and tibias) and the spleen. The FACS analysis results are representative of 4 biological replicates. (C) Representative confocal images of GFP- and PKH-positive cells are shown. The images are merged from three different channels (DAPI, RFP and GFP) to better visualize and contrast human cells in a murine microenvironment. The arrows indicate GFP- and human PKH (RFP)-positive cells, and all cells were stained with the nucleic acid stain DAPI. Scale bar, 75 µm.*p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).

Mentions: We analyzed the colony-forming abilities of PAX5− MCL cells using phytohemagglutinin leukocyte conditioned medium (PHA-LCM), which has been used to evaluate the stem-like properties of myeloma.25 PAX5 silencing significantly increased the number of MCL colony-forming units compared to the control treatment (Figure 2a).


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− cells demonstrate increased colony formation and PKH26+ cell retention in xenografted mice(A) PAX5− cells (5×103) were seeded in PHA-LCM, and colonies were scored at day 7. Only colonies containing > 50 cells were assigned a positive score. Inset: PAX5− cells also formed larger colonies in PHA-LCM, as visualized using a light microscope with a 20x objective. Each value represents the mean number of counted colonies ± S.D. (n=3). Lower panel: The counted colonies of SP53 cells (Left) and Jeko cells (Right) were represented in a graph. PAX5− cells exhibited higher mean numbers of colonies formed ± S.D. (n=3). (B) PAX5− cells displayed increased PKH26+ cell retention in xenografted mice. PKH26+ GFP+ PAX5− cells (1×106) were I.V. transferred to NOD/SCID mice. The mice were sacrificed 48 hours after injection, and cells were collected from bone marrow (femurs and tibias) and the spleen. The FACS analysis results are representative of 4 biological replicates. (C) Representative confocal images of GFP- and PKH-positive cells are shown. The images are merged from three different channels (DAPI, RFP and GFP) to better visualize and contrast human cells in a murine microenvironment. The arrows indicate GFP- and human PKH (RFP)-positive cells, and all cells were stained with the nucleic acid stain DAPI. Scale bar, 75 µm.*p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).
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Figure 2: PAX5− cells demonstrate increased colony formation and PKH26+ cell retention in xenografted mice(A) PAX5− cells (5×103) were seeded in PHA-LCM, and colonies were scored at day 7. Only colonies containing > 50 cells were assigned a positive score. Inset: PAX5− cells also formed larger colonies in PHA-LCM, as visualized using a light microscope with a 20x objective. Each value represents the mean number of counted colonies ± S.D. (n=3). Lower panel: The counted colonies of SP53 cells (Left) and Jeko cells (Right) were represented in a graph. PAX5− cells exhibited higher mean numbers of colonies formed ± S.D. (n=3). (B) PAX5− cells displayed increased PKH26+ cell retention in xenografted mice. PKH26+ GFP+ PAX5− cells (1×106) were I.V. transferred to NOD/SCID mice. The mice were sacrificed 48 hours after injection, and cells were collected from bone marrow (femurs and tibias) and the spleen. The FACS analysis results are representative of 4 biological replicates. (C) Representative confocal images of GFP- and PKH-positive cells are shown. The images are merged from three different channels (DAPI, RFP and GFP) to better visualize and contrast human cells in a murine microenvironment. The arrows indicate GFP- and human PKH (RFP)-positive cells, and all cells were stained with the nucleic acid stain DAPI. Scale bar, 75 µm.*p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).
Mentions: We analyzed the colony-forming abilities of PAX5− MCL cells using phytohemagglutinin leukocyte conditioned medium (PHA-LCM), which has been used to evaluate the stem-like properties of myeloma.25 PAX5 silencing significantly increased the number of MCL colony-forming units compared to the control treatment (Figure 2a).

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