Loss of Rad51c leads to embryonic lethality and modulation of Trp53-dependent tumorigenesis in mice

Abstract

RecA/Rad51 protein family members (Rad51, Rad51b, Rad51c, Rad51d, Xrcc2, and Xrcc3) are essential for DNA repair by homologous recombination, and their role in cancers has been anticipated. Here we provide the first direct evidence for a tumor suppressor function for a member of the Rad51 family. We show that Rad51c deficiency leads to early embryonic lethality, which can be delayed on a Trp53-null background. To uncover the role of Rad51c in tumorigenesis, we have exploited the fact that Rad51c and Trp53 are both closely located on the mouse chromosome 11. We have generated double heterozygous (DH) mice carrying mutant alleles of both genes either on different (DH-trans) or on the same chromosome (DH-cis), the latter allowing for a deletion of wild-type alleles of both genes by loss of heterozygosity. DH-trans mice, in contrast to DH-cis, developed tumors with latency and spectrum similar to Trp53 heterozygous mice. Strikingly, Rad51c mutation in DH-cis mice promoted the development of tumors of specialized sebaceous glands and suppressed tumors characteristic of Trp53 mutation. In addition, DH-cis females developed tumors significantly earlier than any other group.

Figure 1. Mouse Rad51c gene targeting

(A) Scheme illustrating the gene targeting strategy to generate a Rad51c-null allele. Rad51c exons are indicated as boxes with corresponding numbers. Restriction sites are labeled as S for SalI, H - HpaI, R - EcoRI, and K - KpnI. F1, F2, and R1 designate location and direction of primers used for PCR-genotyping. HpaI restriction fragments detected by Southern analysis with the internal probe b are indicated as blue lines under each allele. A frt-loxP-PGK-EM7-neomycin-bp(A)-frt-loxP cassette was targeted into the first intron of Rad51c (at genomic location chr11: 87217150) and a single loxP site was inserted the third intron (at genomic location Chr11: 87214383) (B) Depiction of exon structure of the Rad51c transcript and the corresponding protein with functionally important regions. N-terminal domain is shown in green and C-terminal domain in shown in blue. (C) Genotyping by Southern blot showing four different genotypes with allele sizes labeled at the right. (D) Examples of genotyping using PCR primers shown in (A).

Figure 2. Rad51c^ko/ko mice die during early embryogenesis

(A) Early postimplantation embryos sectioned with the deciduas and stained with hematoxylin and eosin. Top left, developmentally normal control E5.5 embrys reveals equally developed embryonic (e) and extraembryonic (x) tissues with a proamniotic cavity (p) clearly visible. Top right, mutant embryo demonstrates reduced embryonic tissues (e) without the cavity. At E6.5, development of embryonic tissues of the mutant embryo (bottom right) continues to lag behind compared with control embryos (Bottom left). (B) control (top left) and mutant (top right) embryos at E7.5. Primitive streak (s) is observed in control embryo. Morphology of control (bottom left) and mutant (bottom right) embryos at E8.5. x and e, as above; y, yolk sac. (C) Embryonic tissues in mutant embryo at E8.5 reveal active proliferation as evidenced by BrdU staining (top right) and increased apoptosis detected by TUNEL assay (bottom right) compared with control littermates (top and bottom left, respectively). n, neural folds; m, somites; a, amnion; sv, sinus venosus; xc, extraembryonic coelomic cavity; ch, prospective chorion; cc, ectoplacental cavity. Scale bar corresponds to 100 μm in A, and to 500 μm in B-D. (D) Partial rescue of the Rad51c-null phenotype on Trp53-deficient genetic background. Rad51c^ko/ko; Trp53^ko/ko embryo at E10.5 (right) has almost normal morphology but smaller than a control Rad51c^ko/+; Trp53^ko/+ littermate (left). Notice truncated caudal region and unclosed head folds in the mutant embryo (arrowhead). Mutant embryo shows apparently normal looking heart (h).

Figure 3. Interaction between Rad51c and Trp53 in mouse tumorigenesis

(A) Schematic illustration of the five genotypic mouse cohorts used in the study. Vertical lines with a circle at the top indicate mouse chromosome 11 with a centromere. Open box specifies a mutant Rad51c allele. Closed box represents a mutant Trp53 allele. (B and C) Kaplan-Meier plot showing tumor-free survival for each group separately for females (B) and males (C). Statistical evaluation of these data is shown in Table 1. (D) Southern blot analysis of tumor tissues for LOH at Trp53 locus (upper panel) and Rad51c locus (lower panel). Animal genotype is indicated at the bottom. “T” marks tumor samples T1, sarcoma, NOS, muscle; T2, B-cell Lymphoma; T3a, Pituitary adenoma; T3b Hemangiosarcoma; T4, sarcoma, NOS, muscle; T5 rhabdomyosarcom, muscle; T6, sarcoma, NOS, muscle; T7, mammary adenoma; T8, rhabdomyosarcoma, muscle; T9a, rhabdomyosarcoma, muscle; T9b, rhabdomyosarcoma, muscle. T10, cholangiosarcoma,liver; T11a, osteosarcoma, vertebra; T11b, myoepitheliaoma, salivary gland; Letters a and b after the tumor number, indicate that the tumors are from the same animal. Control lanes show tail DNA samples for the tumor samples in the following lane. The only tumor from Rad51c^ko/+ animal showing LOH for Rad51c and another tumor from DH-cis animal showing LOH for Trp53 but not Rad51c is indicated with an asterisk (*) next to tumor number. Faint wild-type bands observed in LOH samples are likely be due to the presence of contaminating normal infiltrating inflammatory cells. Numbers at the bottom of each panel indicate the number of samples showing LOH relative to the total number of tumor samples tested. n.a., not applicable.

Figure 4. Loss of Rad51c results in proliferation defect and genomic instability in MEFs

(A) Deletion of Rad51c induces growth arrest in primary MEFs. Wild-type, Rad51c^neo/+ or Rad51c^neo/neo MEFs have been infected with adenovirus carrying Cre-recombinase and proliferation has been tested using the 3T3 protocol. (B) Sensitivity of MEFs isolated from E10.5 mouse embryos to mitomycin C (MMC). (C) Rad51c-null MEFs do not form Rad51 foci 6 hours after γ-irradiation (10 Gy). Merged images are shown with Rad51 stained green and γH2AX in red. Genotype of the cell is indicated at the bottom of each image. (D) Rad51c-deficient cells accumulate various chromosomal aberrations especially after MMC treatment. The number for each aberration type is depicted proportionally in a stacked bar graph for each cell line. Genotype, passage number (P) and treatment (+ or − MMC) are indicated below each bar.