Calmodulin protects cells from death under normal growth conditions and mitogenic starvation but plays a mediating role in cell death upon B-cell receptor stimulation

Abstract

Calmodulin (CaM) is the main intracellular Ca2+ sensor protein responsible for mediating Ca2+ triggered processes. Chicken DT40 lymphoma B cells express CaM from the two genes, CaMI and CaMII. Here we report the phenotypes of DT40 cells with the CaMII gene knocked out. The disruption of the CaMII gene causes the intracellular CaM level to decrease by 60%. CaMII-/- cells grow more slowly and die more frequently as compared to wild type (wt) cells but do not exhibit significant differences in their cell cycle profile. Both phenotypes are more pronounced at reduced serum concentrations. Upon stimulation of the B-cell receptor (BCR), the resting Ca2+ levels remain elevated after the initial transient in CaMII-/- cells. Despite higher Ca2+ resting levels, the CaMII-/- cells are partially protected from BCR induced apoptosis indicating that CaM plays a dual role in apoptotic processes.

Figure 1

Homologous recombination at the CaMII locus. Partial restriction map of the chicken CaMII gene (a), Targeting constructs (b), Structure of the disrupted alleles (c), and Southern blot analysis of genomic DNAs (d). Exon 1 to exon 6 are shown by boxes. The restriction endonuclease cleavage sites are abbreviated as B=BamHI; E=EcoRI; Ev=EcoRV; X=XhoI. Genomic DNA was prepared from wild type (+/+, lane 1), HisD-targeted (±, lane 2) and HisD/Neo-targeted (−/−, lane 3) cells and digested with EcoRI. A 3′ cDNA coding region of CaMII (600 bp PstI fragment32) was used as a probe.

Figure 2

Northern blot analysis of CaMII transcripts (a) and immunodetection of CaM levels (b,c). (a) For Northern blot analysis 20 µg total RNA was separated on a 1·2% agarose gel containing 6% formaldehyde, blotted and probed with chicken CaMII cDNA. (b) For immunoblot analysis of total lysates 10 µg of total protein was separated by 12% SDS–PAGE and probed with anti-CaM antibody. C CaM signals were quantified with a Storm PhosphoImager. Columns represent the mean (± SD, n = 3) relative intracellular CaM levels.

Figure 3

Analysis of growth rate (a), cell cycle (b) and death rate (c) under normal growth conditions. (a) For determination of cell growth, cells were seeded at a density of 1 × 10⁴ cells/ml and counted in 6 hr intervals using a haemocytometer. The generation time was calculated from the slope of the logarithmic growth curve during exponential growth. Columns show the mean (± SD, n = 6) generation time. *P < 0·025 (8-3/wt), **P < 0·005 (8-7/wt) by Student's t-test. (b) For cell cycle analysis cells 1 × 10⁶ grown in normal medium were fixed in 70% ethanol/PBS and stained with propidium iodide. Columns show the mean (± SD, n = 6) percentage of cells found in a particular cell cycle phase. *P < 0·005 (8-3/wt at S phase; 8-3/wt and 8-7/wt at G2M phase), **P < 0·0005 (8-7/wt at S phase) by Student's t-test. (c) For viability analysis cells 2 × 10⁵ were stained with merocyanine and analysed by FACS. Columns show the mean (± SD, n = 11) percentage of dead cells found in the analysed populations. *P < 0·0005 (8-3/wt; 8-7/wt) by Student's t-test.

Figure 4

Analysis of growth (a), cell cycle (b) and death rate (c) at reduced serum concentration. Cell samples for (a), (b) and (c) were taken from the same culture dishes. (a) For growth measurement cells (1 × 10⁵) were incubated in the indicated media for 24 hr at 40° and cell numbers were assessed with a haemocytometer. The data points represent the mean (± SD, n = 3) cell number in percentage of the control at 10% FCS. *P < 0·005 (8-3/wt; 8-7/wt) by Student's t-test (FCS, fetal calf serum; CS chicken serum). (b) DNA profiles were determined by propidium iodide staining and FACS analysis. Data points represent the mean (± SD, n = 4) percentage of cells in the given cell cycle phases. The S phase was the only cell cycle phase that changed significantly in both mutant cell lines (8-3 P < 0·005; 8-7 P < 0·05 by Student's t-test). (c) Cell viability was assessed by merocyanine staining and FACS. The data points represent the mean (± SD, n = 3) viability. *P < 0·005 (8-3/wt; 8-7/wt at 0·1/1% FCS/CS) by Student's t-test.

Figure 5

Analysis of intracellular Ca²⁺ transients after BCR crosslinking. Cells (5 × 10⁵/ml) were loaded with Fura-2AM for 30 min at 40° and followed by stimulation with the anti-chicken IgM antibody (clone M4). The Ca²⁺ levels were recorded by measuring the emission ratio at 510 nm of the two excitation wavelengths 340 nm and 380 nm measured. The columns represent the mean difference (n = 15) between the 340/380 ratios at 400 seconds and at t = 0.

Figure 6

Analysis of BCR induced cell cycle arrest (a) and cell death (b). Cells (1 × 10⁶/ml) were stimulated with 4 µg/ml anti-sIgM antibody for 24 hr. (a) For cell cycle analysis cells were fixed in 70% ethanol/PBS and stained with propidium iodide prior to FACS analysis. The columns represent the mean (± SD, n = 3) percentage of cells in a particular cell cycle compartment (P < 0·005 wt, 8-3 and 8-7 M4 stimulated/non-stimulated in S and G2/M phases; P < 0·05 8-3/wt; 8-7/wt in G2 and M phases after M4 stimulation, all other differences between wt and CaMII ko cells after M4 stimulation were not significant (P > 0·05). (b) Cell death was analysed by staining the cells with merocyanine before FACS analysis. Death induction was calculated by subtracting the percentages of dead cells in the control from the stimulated populations. The columns represent the mean (± SD, n = 5) percentage of induced cell death * P < 0·005 (8-3/wt; 8-7/wt) by Student's t-test.

Figure 7

Tyrosine phosphorylation in wild type, CaMII(−/−) and CaM(+/−) DT40 cells following activation with anti-IgM (M4). At the indicated times following the addition of M4 (4 µg/ml), whole-cell lysates prepared from 2·5 × 10⁶ cells were loaded onto an 8% SDS–PAGE gel. After transfer to nitrocellulose, the filter was incubated with antiphosphotyrosine mAb 4G10.