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Establishment and Characterization of PCL12, a Novel CD5+ Chronic Lymphocytic Leukaemia Cell Line.

Agathangelidis A, Scarfò L, Barbaglio F, Apollonio B, Bertilaccio MT, Ranghetti P, Ponzoni M, Leone G, De Pascali V, Pecciarini L, Ghia P, Caligaris-Cappio F, Scielzo C - PLoS ONE (2015)

Bottom Line: PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters.Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice.PCL12 represents a suitable preclinical model for testing pharmacological agents.

View Article: PubMed Central - PubMed

Affiliation: IRCCS San Raffaele Scientific Institute, Division of Experimental Oncology, Unit of Lymphoid Malignancies, Milano, Italy; IRCCS San Raffaele Scientific Institute, Division of Experimental Oncology, Unit of B Cell Neoplasia, Milano, Italy.

ABSTRACT
Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the "original" clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents.

No MeSH data available.


Related in: MedlinePlus

(a) PCL12 cells images acquired with Nikon TS100 microscope after 4 weeks of culture (20x magnification objective in the left panel), dark arrow indicating nurse like cells, and 6 weeks of culture (4x magnification objective on the right).(b) Haematoxylin and Eosin staining (c) WB analysis for LMP1 protein on PCL12 cells, Positive control JY cell line and negative RL cell line. (d) PCL12 immunophenotype dot plot acquired by Flow cytometry.
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pone.0130195.g001: (a) PCL12 cells images acquired with Nikon TS100 microscope after 4 weeks of culture (20x magnification objective in the left panel), dark arrow indicating nurse like cells, and 6 weeks of culture (4x magnification objective on the right).(b) Haematoxylin and Eosin staining (c) WB analysis for LMP1 protein on PCL12 cells, Positive control JY cell line and negative RL cell line. (d) PCL12 immunophenotype dot plot acquired by Flow cytometry.

Mentions: Peripheral blood mononuclear cells (PBMC) were obtained by gradient separation after admission. We seeded 3x106 total PBMC and 3x106 purified CD19+ cells in RPM1 medium supplemented with 10% FBS and 15 mg/ml Gentamicin in 25 cm2 filtered flasks and followed cell growth over time. While purified CD19+ cells died after 2 weeks of culture, in the total PBMC cultures we observed, after 4 weeks the persistence of mononuclear cells that tended to form clusters in close contact with adherent cells, the latter being likely nurse-like cells [19], (Fig 1A left panel). After 2 additional weeks, cells started to grow in suspension forming clumps. These cells were CD19+CD5+ (98%) (Fig 1D) and they rapidly expanded when transferred to a new flask (Fig 1A right panel). Cells have been maintained in culture for over 1 year without any difficulty. The cell morphology was evaluated with Haematoxylin and Eosin staining (Fig 1B). We tested the cell line for the presence of EBV viral proteins and we observed high LMPI protein expression (Fig 1C).


Establishment and Characterization of PCL12, a Novel CD5+ Chronic Lymphocytic Leukaemia Cell Line.

Agathangelidis A, Scarfò L, Barbaglio F, Apollonio B, Bertilaccio MT, Ranghetti P, Ponzoni M, Leone G, De Pascali V, Pecciarini L, Ghia P, Caligaris-Cappio F, Scielzo C - PLoS ONE (2015)

(a) PCL12 cells images acquired with Nikon TS100 microscope after 4 weeks of culture (20x magnification objective in the left panel), dark arrow indicating nurse like cells, and 6 weeks of culture (4x magnification objective on the right).(b) Haematoxylin and Eosin staining (c) WB analysis for LMP1 protein on PCL12 cells, Positive control JY cell line and negative RL cell line. (d) PCL12 immunophenotype dot plot acquired by Flow cytometry.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481539&req=5

pone.0130195.g001: (a) PCL12 cells images acquired with Nikon TS100 microscope after 4 weeks of culture (20x magnification objective in the left panel), dark arrow indicating nurse like cells, and 6 weeks of culture (4x magnification objective on the right).(b) Haematoxylin and Eosin staining (c) WB analysis for LMP1 protein on PCL12 cells, Positive control JY cell line and negative RL cell line. (d) PCL12 immunophenotype dot plot acquired by Flow cytometry.
Mentions: Peripheral blood mononuclear cells (PBMC) were obtained by gradient separation after admission. We seeded 3x106 total PBMC and 3x106 purified CD19+ cells in RPM1 medium supplemented with 10% FBS and 15 mg/ml Gentamicin in 25 cm2 filtered flasks and followed cell growth over time. While purified CD19+ cells died after 2 weeks of culture, in the total PBMC cultures we observed, after 4 weeks the persistence of mononuclear cells that tended to form clusters in close contact with adherent cells, the latter being likely nurse-like cells [19], (Fig 1A left panel). After 2 additional weeks, cells started to grow in suspension forming clumps. These cells were CD19+CD5+ (98%) (Fig 1D) and they rapidly expanded when transferred to a new flask (Fig 1A right panel). Cells have been maintained in culture for over 1 year without any difficulty. The cell morphology was evaluated with Haematoxylin and Eosin staining (Fig 1B). We tested the cell line for the presence of EBV viral proteins and we observed high LMPI protein expression (Fig 1C).

Bottom Line: PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters.Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice.PCL12 represents a suitable preclinical model for testing pharmacological agents.

View Article: PubMed Central - PubMed

Affiliation: IRCCS San Raffaele Scientific Institute, Division of Experimental Oncology, Unit of Lymphoid Malignancies, Milano, Italy; IRCCS San Raffaele Scientific Institute, Division of Experimental Oncology, Unit of B Cell Neoplasia, Milano, Italy.

ABSTRACT
Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the "original" clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents.

No MeSH data available.


Related in: MedlinePlus