Tsg101 is essential for cell growth, proliferation, and cell survival of embryonic and adult tissues

Abstract

Tumor susceptibility gene 101 (Tsg101) was identified in a random mutagenesis screen for potential tumor suppressors in NIH 3T3 cells. Altered transcripts of this gene have been detected in sporadic breast cancers and many other human malignancies. However, the involvement of this gene in neoplastic transformation and tumorigenesis is still elusive. Using gene targeting, we generated genetically engineered mice with a floxed allele of Tsg101. We investigated essential functions of this gene in vivo and examined whether the loss of function of Tsg101 results in tumorigenesis. Conventional knockout mice were generated through Cre-mediated excision of the first coding exon in the germ line of mouse mammary tumor virus (MMTV)-Cre transgenic mice. The complete ablation of Tsg101 in the developing embryo resulted in death around implantation. In contrast, mammary gland-specific knockout mice developed normally but were unable to nurse their young as a result of impaired mammogenesis during late pregnancy. Neither heterozygous null mutants nor somatic knockout mice developed mammary tumors after a latency of 2 years. The Cre-mediated deletion of Tsg101 in primary cells demonstrated that this gene is essential for the growth, proliferation, and survival of mammary epithelial cells. In summary, our results suggest that Tsg101 is required for normal cell function of embryonic and adult tissues but that this gene is not a tumor suppressor for sporadic forms of breast cancer.

FIG. 1.

Targeted deletion of the Tsg101 gene. (A) Targeting strategy to flank the promoter and first coding exon of Tsg101 with loxP sites. The conditional knockout (floxed) allele contains a selectable marker cassette 3 kb upstream and a third loxP site 230 bp downstream of the first coding exon. (B) Southern blot analysis of genomic DNA of mice carrying either two floxed alleles (fl/fl), one floxed and one wild-type allele (fl/+), or two wild-type alleles (+/+). The 11.5-kb EcoRI restriction fragment represents the wild-type allele, whereas homozygous mutants carry only a 4-kb fragment. (C) Cre-mediated deletion of the Tsg101 gene. (D) Southern blot to verify the correct deletion event illustrated in panel C. Genomic DNA was isolated from tail biopsy specimens from transgenic mice carrying the MMTV-Cre transgene and either two Tsg101 wild-type alleles (Tsg101^+/+) or one floxed and one wild-type allele (Tsg101^fl/+). The 2.1-kb XbaI fragment represents the recombined Tsg101 null allele that is present only when a floxed allele is subject to Cre-mediated recombination. Both the wild-type and unrecombined floxed allele are represented by a 2.7-kb XbaI restriction fragment. R, EcoRI; H, HindIII; N, NotI; X, XbaI; tk, thymidine kinase.

FIG. 2.

Examination of Tsg101-deficient embryos. (A) Tsg101 knockout embryos are phenotypically indistinguishable from their wild-type littermates at E3.5. PCR was used to verify the absence of the wild-type allele in these embryos. B. Deciduae at E6.5 from two Tsg101 heterozygous knockout crosses. At this stage of development, Tsg101 knockouts could not be identified. The abnormal-looking deciduum (arrow) contained a heterozygous mutant.

FIG. 3.

Analysis of lactation (A) and histological examination of mammary glands (B) from tissue-specific Tsg101 knockout mice (WAP-Cre Tsg101^fl/fl) and their controls (Tsg101^fl/fl). (A) A mouse is defined as lactating if she can support at least one pup for 12 days. (B) Mammary gland whole mounts were stained in carmine alum (magnification, ×40) and subsequently embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) (magnification, ×100).

FIG. 4.

Southern blot (A and B) and Northern blot (C and D) analyses to determine the recombination efficiency and expression levels of Tsg101 in the mammary glands of Tsg101 conditional mutants (WAP-Cre Tsg101^fl/fl) and their controls (Tsg101^fl/fl or WAP-Cre Tsg101^fl/+). (A and B) The 2.1-kb XbaI fragment represents the recombined Tsg101 null allele. The unrecombined floxed or wild-type alleles are 2.7 kb (see Fig. 1). (A and C) Recombination efficiency and Tsg101 mRNA expression levels in mammary tissues from mice during late pregnancy (day 18 of gestation) and around parturition (16 h postpartum). (B and D) Southern and Northern analyses of mammary glands from lactating mice at 1, 3, and 12 days of lactation. Note the decline in the number of Tsg101-deficient cells in conditional knockout animals that are able to establish lactation (compare lanes fl/fl yes for 18 days and 16 h in panel A with lanes fl/fl yes for 72 h and fl/fl yes for 12 days [3 lanes] in panel B).

FIG. 5.

Detection of proliferating (arrows in panels B to D) and apoptotic (arrows in panels E to H) cells at various stages of mammary development in Tsg101 mammary-specific knockout mice (WAP-Cre Tsg101^fl/fl B, D, and F to H) and their controls (Tsg101^fl/fl [A, C, and E]). (A and B) 17 days of gestation (magnification, ×400). (C to H) Shortly after parturition (magnification, ×400). Proliferating cells were detected using BrdU labeling, whereas apoptotic cells were identified by immunohistochemistry using the M30 CytoDEATH antibody. Slides were counterstained with hematoxylin. Note the larger number of dying cells in less organized or collapsed alveolar structures (F to H).

FIG. 6.

Tsg101 deletion in primary epithelial cultures. (A) Experimental design. (B) Pan-cytokeratin staining to verify the presence of epithelial cells (magnification, ×400). (C) Tsg101 protein expression in uninfected (no virus) and Cre expressing (pBabe-Cre) cultures. The blot was reprobed with an antibody against β-actin to verify equal loading between lanes. (D) Declining amounts of viable cells in Tsg101-deficient cultures (Tsg101^fl/fl; pBabe-Cre) between 3 and 7 days after Cre expression. Tsg101^fl/fl cultures infected with pBabe or wild-type cells expressing Cre (Tsg101^+/+; pBabe-Cre) served as controls.

FIG. 7.

Immunocytochemistry for PCNA (A) and cyclin A (B) in Tsg101-deficient cultures (Tsg101^fl/fl; pBabe-Cre) and their controls (Tsg101^fl/fl). Nuclei were counterstained with DAPI. Note the presence of replicating cells and the expression and nuclear accumulation of cyclin A in the controls, which indicates that these cells, in contrast to the Tsg101 knockouts, progress normally into S phase.