The promyelocytic leukemia zinc-finger gene, PLZF, is frequently downregulated in malignant mesothelioma cells and contributes to cell survival

Abstract

DNA copy number analysis was performed, using single-nucleotide polymorphism mapping arrays, to fine map genomic imbalances in human malignant mesothelioma (MM) cell lines derived from primary tumors. Chromosomal losses accounted for the majority of genomic imbalances. All 22 cell lines examined showed homozygous deletions of 9p21.3, centering at the CDKN2A/ARF and CDKN2B loci. Other commonly underrepresented segments included 1p36, 1p22, 3p21-22, 4q13, 4q34, 11q23, 13q12-13, 14q32, 15q15, 18q12, and 22q12, each observed in 55-90% of cell lines. Focal deletions of 11q23 encompassed the transcriptional repressor gene promyelocytic leukemia zinc finger (PLZF), which was validated by analysis of genomic DNA using real-time polymerase chain reaction (PCR). Semi-quantitative RT-PCR and immunoblot analysis revealed that PLZF is greatly downregulated in MM cell lines compared with non-malignant mesothelial cells. Ectopic expression of PLZF in PLZF-deficient MM cells resulted in decreased cell viability, reduced colony formation, as well as increased apoptosis, the latter based on results of various cell death assays and the observation of increased cleavage of caspase 3, PARP, and Mcl-1. These data indicate that deletions of PLZF are a common occurrence in MM and that downregulation of PLZF may contribute to MM pathogenesis by promoting cell survival.

Figure 1

A) DNA copy number analysis profile of the entire genome of a representative MM cell line showing multiple alterations, including nearly all of the recurrent chromosomal deletions (red arrows) seen in the overall series. The 3p amplicon (green arrow) is notable in that the boundary between the proximal amplified segment and the more distal deletion resides within the FHIT gene located at a fragile site in 3p14.2. B) Schematic summary of CNAs observed in the entire set of MM cell lines highlighting common regions of copy number losses (red arrows) in multiple chromosomes, including 1p, 3p, 4p/q, 6q, 9p, 11q, 13q, 14q, 15q, 18q, and 22q. Note that all 22 cell lines showed homozygous deletions of 9p21.3, centering at the CDKN2A/ARF and CDKN2B loci.

Figure 2

A) DNA copy number analysis profiles of chromosome 11 in three MM cell lines. Losses overlap in 11q23.2-23.3, including one cell line with a focal deletion encompassing the PLZF gene. B) Upper panel: Semi-quantitative RT-PCR showing downregulation of PLZF in 11 MM cell lines compared to the expression observed in control mesothelial cells, LP9 and LP9/TERT-1. Lower panel: Immunoblot analysis demonstrating downregulation of PLZF protein in MM cells compared to control mesothelial cells.

Figure 3

Re-expression of PLZF in MM cells. A) Transfection of PLZF expression construct in four MM cell lines resulted in diminished cell proliferation, based on MTS assay 4 days after transfection using Amaxa Nucleofector. B) Representative clonogenic assay showing decreased colony formation in MM cells transfected with PLZF expression construct compared to cells transfected with empty vector. Cells were selected for 7 d in puromycin as described in the Materials and Methods. Quantitation of the clonogenic assays showed statistically significant differences (p < 0.05, 2-tailed Student t-test).

Figure 4

PLZF re-expression in MM cells leads to increased apoptosis. A) Transfection of PLZF expression construct in four MM cell lines resulted in increased apoptosis, demonstrated by use of the Cell Death Detection ELISA plus assay 24 and 48 hours after transfection using Amaxa Nucleofector reagent. The results were statistically different (p < 0.05, 2-tailed Student t-test). B) Increase in sub-G1 percentage 48 hours after PLZF transfection. C) Western blot analysis demonstrating an increase in the pro-apoptotic Mcl-1 cleaved fragment (Mcl-1*) in MM cells transfected with PLZF expression construct compared to that observed in cells transfected with empty vector. Increased cleaved caspase-3 and cleaved PARP, indicative of increased apoptosis, were also observed. Immunoblotting for other pro-apoptotic or pro-survival factors shown did not exhibit any consistent expression changes related to expression of PZLF. Cells were harvested 48 hours after transfection, and lysates were subjected to immunoblot analysis. Equal amounts (30 µg) of protein were subjected to SDS-PAGE, blotted, and incubated with various antibodies shown.

Figure 4

PLZF re-expression in MM cells leads to increased apoptosis. A) Transfection of PLZF expression construct in four MM cell lines resulted in increased apoptosis, demonstrated by use of the Cell Death Detection ELISA plus assay 24 and 48 hours after transfection using Amaxa Nucleofector reagent. The results were statistically different (p < 0.05, 2-tailed Student t-test). B) Increase in sub-G1 percentage 48 hours after PLZF transfection. C) Western blot analysis demonstrating an increase in the pro-apoptotic Mcl-1 cleaved fragment (Mcl-1*) in MM cells transfected with PLZF expression construct compared to that observed in cells transfected with empty vector. Increased cleaved caspase-3 and cleaved PARP, indicative of increased apoptosis, were also observed. Immunoblotting for other pro-apoptotic or pro-survival factors shown did not exhibit any consistent expression changes related to expression of PZLF. Cells were harvested 48 hours after transfection, and lysates were subjected to immunoblot analysis. Equal amounts (30 µg) of protein were subjected to SDS-PAGE, blotted, and incubated with various antibodies shown.