Targeted deletion of the S-phase-specific Myc antagonist Mad3 sensitizes neuronal and lymphoid cells to radiation-induced apoptosis - PubMed (original) (raw)

Targeted deletion of the S-phase-specific Myc antagonist Mad3 sensitizes neuronal and lymphoid cells to radiation-induced apoptosis

C Quéva et al. Mol Cell Biol. 2001 Feb.

Abstract

The Mad family comprises four basic-helix-loop-helix/leucine zipper proteins, Mad1, Mxi1, Mad3, and Mad4, which heterodimerize with Max and function as transcriptional repressors. The balance between Myc-Max and Mad-Max complexes has been postulated to influence cell proliferation and differentiation. The expression patterns of Mad family genes are complex, but in general, the induction of most family members is linked to cell cycle exit and differentiation. The expression pattern of mad3 is unusual in that mad3 mRNA and protein were found to be restricted to proliferating cells prior to differentiation. We show here that during murine development mad3 is specifically expressed in the S phase of the cell cycle in neuronal progenitor cells that are committed to differentiation. To investigate mad3 function, we disrupted the mad3 gene by homologous recombination in mice. No defect in cell cycle exit and differentiation could be detected in mad3 homozygous mutant mice. However, upon gamma irradiation, increased cell death of thymocytes and neural progenitor cells was observed, implicating mad3 in the regulation of the cellular response to DNA damage.

PubMed Disclaimer

Figures

FIG. 1

Expression of mad3 in neural progenitor cells. (A) Caudal section of a day 11.5 p.c. mouse embryo. The neural tube (nt) shows little sign of differentiation, as evidenced by the absence of the IZ, and consists primarily of proliferating neural progenitors. Brown-stained nuclei indicate cells that have incorporated BrdU for 1 h and are in the S phase of the cell cycle. The section was also hybridized with a mad3 antisense riboprobe. mad3 signal is realtively weakly detected in the proliferating neural progenitors but can be seen in tissue surrounding the neural tube (see panel D). (B) The expression of mad3 is restricted to the periphery of the VZ. No signal is detected in the IZ with the mad3 probe on this day 10.5 p.c. transverse section. (C) Transverse section though the neural tube in a truncal location at day 11.5 p.c. Cells in S phase of the cell cycle are labeled with BrdU (1 h) (brown stain). In the neural tube, the labeled nuclei are localized at the periphery of the VZ. The IZ contains the postmitotic neurons unlabeled with BrdU. Occasional endothelial cells are BrdU labeled. (D) Colocalization of BrdU-positive cells and mad3 in situ hybridization signal. Shown is a transverse section of the neural tube at day 11.5 p.c. processed for immunocytochemistry for the detection of BrdU-incorporating cells and for in situ hybridization to detect mad3. The nuclei at the periphery of the VZ are labeled by BrdU and are also highlighted by the mad3 hybridization signal. Doubly labeled cells can also be observed in the dorsolateral part of the neural tube and are likely to be neural crest cells (arrows). (E) Higher magnification of the VZ pictured in panel D, showing the juxtaposition of mad3 expression and the BrdU-labeled neural progenitors.

FIG. 2

Targeted disruption of the mad3 gene. (A) Map of the mad3 locus, showing the exon-intron structure of the gene. From 5′ to 3′, the exons encode the SID (E1), the conserved HGYAS motif (E2), the basic region (E3), the HLH (E4), the LZ (E5), and the C-terminal segment (E6), respectively. The targeting construct comprised the pPGK_neo_bpAPGK_dta_bpA cassette flanked by an 845-bp _Nhe_I fragment and a 5.8-kb _Bam_HI-_Sac_II fragment for recombination arms. Negative selection was provided by PGK_dta_bpA. Homologous recombination yielding to the targeted locus was selected by PCR using the primers M3133 and YZ29 (arrows) and later confirmed by Southern blotting using probes A and B. (B) Genomic Southern blot analysis confirming the correct 5′ (probe A) and 3′ (probe B) junctions. (C) PCR genotyping of mad3+/+, mad3+/−, and _mad3_−/− mice using the primers depicted as arrowheads in panel A. (D) Detection of mad3 by RT-PCR. In embryos obtained from the breeding of mad3+/− mice, RT-PCR confirmed the absence of mad3 expression in _mad3_−/− mice and demonstrated that a functionally null mutation was generated. Amplification of the S16 mRNA was used as a control for efficient cDNA synthesis. wt, wild type.

FIG. 3

Increased sensitivity of _mad3_−/− thymocytes to gamma irradiation. (A) Thymocytes from mad3+/+ and _mad3_−/− mice were left untreated (medium), treated with gamma irradiation (200 rads), and then incubated in tissue culture medium containing no addition (medium or 200 rads), PMA (2 ng/ml) (PMA), dexamethasone (1 μM) (DEX), or ionomycin (1 μg/ml) (ION). Death was determined 24 h after culture by the trypan blue exclusion method. (B) Dose response of mad3+/+ and _mad3_−/− thymocytes to gamma irradiation. Decreased survival of _mad3_−/− thymocytes is observed at 100, 200, 500, and 1,000 rads at 24 h after culture. (C) Determination of the proportion of annexin-positive cells is an independent measurement of the increased apoptosis in _mad3_−/− mice in response to gamma irradiation. Thymocytes harvested from mad3+/+ and _mad3_−/− mice were subjected to 200-rad irradiation. The binding of annexin and the extent of apoptosis were determined by fluorescence-activated cell sorting 24 h after irradiation. (D) Loss of mad3 does not influence cell cycle entry in thymocytes. Thymocytes harvested from mad3+/+ and _mad3_−/− mice were stimulated by concanavalin A. Cell cycle entry was monitored by BrdU incorporation analyzed by fluorescence-activated cell sorting.

FIG. 3

FIG. 4

_mad3_−/− embryos show increased sensitivity to gamma irradiation. Shown are results of TUNEL detection of apoptotic neural precursor cells in transverse sections of mad3+/+ (A) and _mad3_−/− (B) neural tube 3 h after irradiation at 200 rads in utero. The counterstain is hematoxylin. Brown apoptotic nuclei accumulated at the periphery of the VZ and in the lumen of the neural tube more readily in _mad3_-deficient embryos. Cell counting indicated a significant increase in dead cells in the _mad3_−/− neural tubes over those in the wild type (P < 0.05). Wild-type littermate control mice contained an average of 11.8 ± 2.7 apoptotic nuclei while _mad3_-null mice contained an average of 57.5 ± 5.8 apoptotic nuclei in the lumen of the neural tube.

References

1. Amati B, Dalton S, Brooks M W, Littlewood T D, Evan G I, Land H. Transcriptional activation by the human c-Myc oncoprotein in yeast requires interaction with Max. Nature. 1992;359:423–426. - PubMed
1. Amin C, Wagner A J, Hay N. Sequence-specific transcriptional activation by Myc and repression by Max. Mol Cell Biol. 1993;13:383–390. - PMC - PubMed
1. Ayer D E, Eisenman R N. A switch from Myc:Max to Mad:Max heterocomplexes accompanies monocyte/macrophage differentiation. Genes Dev. 1993;7:2110–2119. - PubMed
1. Ayer D E, Kretzner L, Eisenman R N. Mad: a heterodimeric partner for Max that antagonizes Myc transcriptional activity. Cell. 1993;72:211–222. - PubMed
1. Ayer D E, Laherty C D, Lawrence Q A, Armstrong A P, Eisenman R N. Mad proteins contain a dominant transcription repression domain. Mol Cell Biol. 1996;16:5772–5781. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources
Molecular Biology Databases
- GlyGen glycoinformatics resource
- Mouse Genome Informatics (MGI)

Targeted deletion of the S-phase-specific Myc antagonist Mad3 sensitizes neuronal and lymphoid cells to radiation-induced apoptosis - PubMed (original) (raw)