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Previous Article | Next Article 
Molecular and Cellular Biology, January 2003, p. 150-162, Vol. 23, No. 1
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.1.150-162.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805,1 Laboratory of Genetics and Physiology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-08222
Received 31 July 2002/ Returned for modification 9 September 2002/ Accepted 25 September 2002
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ABSTRACT |
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INTRODUCTION |
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Various biological functions of Tsg101 have been postulated from the predicted protein structure, the intracellular localization of Tsg101, the identification of Tsg101 binding proteins, and the deletion of the putative homologous gene Vsp23p in yeast. These functions include a role in ubiquitination (10, 23), transcriptional regulation (8, 36), endosomal trafficking (1, 6), and cell proliferation (37, 40). Although some of these functions have been verified experimentally in cell culture systems, their relevance in vivo during normal development and neoplastic transformation is still elusive. In addition, it has never been confirmed, using a site-directed targeting approach, that the deletion of both endogenous Tsg101 alleles leads to accelerated growth and neoplastic transformation of nonimmortalized cells and to tumorigenesis in vivo. Based on our earlier findings that (i) Tsg101 is expressed in all tissues of the mouse throughout development and (ii) the 5' sequence of this gene has common features of housekeeping gene promoters, we hypothesized that a conventional knockout of this gene would result in early embryonic death (32). Therefore, we have chosen the Cre-loxP technology, which allows the conditional deletion of Tsg101 from any given cell type in genetically engineered mice. In this article, we report that Tsg101 is essential for cell growth, cell survival, and normal function of embryonic and adult tissues. Tsg101-deficient cells exhibited a defect in cell cycle regulation and underwent increased cell death. In contrast to an earlier report (13), a Tsg101 knockout did not result in accelerated cell growth and instant neoplastic transformation. Our findings suggest that a null mutation of Tsg101 is not an initiating event for tumorigenesis.
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MATERIALS AND METHODS |
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Southern and Northern hybridization. Genomic DNA from tissues or cell lines was prepared using standard phenol-chloroform extraction, and 15 µg was digested with EcoRI or XbaI at 37°C overnight. The DNA was separated on a 0.8% agarose gel, blotted onto a nylon membrane (GeneScreen Plus; NEN), and hybridized with a 32P-labeled probe. The 5' diagnostic probe, which was used to screen targeted ES cells, was generated by PCR using the primer set 5'-TTG CTT GTA TGA GTG CAG GTG CC-3' and 5'-TAT GAC CTG CTT CTT GCA AAA GCA G-3'. The 3' internal probe for identification of the various Tsg101 alleles was amplified by PCR using the forward and reverse primers 5'-ATC TTA TCT CCC ATC CTA AGC AGA C-3' and 5'-TCT CTC ACA TCA CTA AAG CTC AAT G-3', respectively. Membranes were washed in 0.1x SSC buffer (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing sodium dodecyl sulfate (SDS) and exposed for 6 h to a Kodak X-Omat-AR film.
The isolation of total RNA from mammary tissue has been described previously (32). A 20-µg portion of total RNA was separated on a 1.5% formaldehyde gel and transferred to a GeneScreen Plus membrane. Tsg101 transcripts were detected by probing the membranes with 32P-labeled full-length mouse Tsg101 cDNA.
Whole-mount analysis of the mammary gland and other histological techniques. The preparation and staining of mammary gland whole mounts and detailed protocols for immunohistochemistry and bromodeoxyuridine (BrdU) labeling of proliferating mammary epithelial cells were described previously (35). For immunohistochemistry, mammary gland tissue was fixed for 5 h in 4% (vol/vol) paraformaldehyde or overnight at 4°C in 10% buffered formalin (Fisher Scientific Co.). The specimens were embedded in paraffin by standard methods. Sections were deparaffinized, rehydrated, and treated with antimasking solution from Vector Laboratories, Inc., as specified by the manufacturer. We used a 1:10 dilution of the M30 CytoDEATH stock solution (see detailed protocol by Roche Molecular Biochemicals) to label apoptotic mammary epithelial cells. The Vectastain Elite ABC and peroxidase substrate kit from Vector Laboratories, Inc., was used to visualize labeled cells. The slides were counterstained with hematoxylin. Immunohistochemical detection of BrdU-labeled nuclei was performed by following the Amersham-Pharmacia-Biotech (RPN202) protocol.
Construction of retroviral expression vectors. The Cre coding sequence from vector pBS185 (a kind gift of B. Sauer, National Institute of Diabetes, Digestive, and Kidney Disease [NIDDK], to L. Hennighausen, NIDDK) was cloned as an XhoI-MluI(blunt) fragment into the EcoRV-XhoI sites of pZero (Invitrogen, Inc.). The pBabe-Cre-puro retroviral vector was constructed by subcloning the Cre recombinase cDNA as an XhoI(blunt)-EcoRI fragment into the BamHI(blunt) and EcoRI sites of pBabe-puro. We have used 293T cells to generate replication-deficient viral particles of pBabe and its Cre-expressing derivative (pBabe-Cre).
Cell culture and immunocytochemistry. Primary mammary epithelial cell cultures were prepared similarly to methods described previously (16, 20). The epithelial cells were maintained in Dulbecco’s minimal essential medium (DMEM)/F12 supplemented with 2% fetal calf serum, 10 µg of insulin per ml, 10 ng of epidermal growth factor per ml, 10 µg of gentamicin per ml, 100 U of penicillin per ml, and 100 µg of streptomycin per ml. For retroviral infection, cells at passages 2 and 3 were plated at a density of 3 x 105 to 4 x 105 cells per 10-cm culture dish. Infection with pBabe-Cre or control constructs lacking the Cre coding sequence (pBabe) was performed in the presence of 10 µg of polybrene (Sigma) per ml. Forty-eight hours later, the cells were selected in complete medium containing 3 to 7 µg of puromycin (Sigma) per ml.
For immunocytochemistry, cells were fixed in 70% ethanol, washed twice in 1x phosphate-buffered saline (PBS) and incubated for 20 min in blocking solution (1x PBS, 1% bovine serum albumin, 0.1% Tween 20). The slides were treated for several hours with a 1:500 to 1:1,000 dilution of the primary antibody in blocking solution (anti-proliferating cell nuclear antigen [PCNA] [PC-10], anti-cyclin A [C-19] [both from Santa Cruz], or anti-pan-cytokeratin [Sigma]). The primary PCNA and anti-pan-cytokeratin antibodies were fluorescein isothiocyanate (FITC)-conjugated. After an additional washing step, cyclin A was visualized with an FITC-conjugated secondary antibody (sc-2012 [Santa Cruz]; 1:1,000 dilution). The slides were washed repeatedly in 1x PBS and mounted with Vectashield containing 1.5 µg of 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc.).
Western blot analysis. Cell pellets were lysed on wet ice for 30 min in 1x PBS-1 % NP-40-0.5% sodium deoxycholate-0.1% SDS, 1 mM phenylmethylsulfonyl fluoride-0.4 U of aprotinin per ml-1 mM NaF-0.1 mM sodium orthovandate. Protein was quantified using a Bradford assay (Pierce). Protein (20 to 50 µg per lane) was resolved by SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes (Invitrogen). The membranes were blocked for 1 h in 1x TBS-0.1% Tween 20-5% dry milk. They were then incubated with primary antibodies in blocking buffer at 4°C overnight, washed three times for 15 min in washing buffer (1x TBS-0.1% Tween 20), and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies in blocking buffer. The membranes were washed again three times in washing buffer and once for 15 min in 1x TBS without Tween 20. Protein bands were detected using the ECL chemiluminescence kit for Western blot analysis (Amersham) as specified by the manufacturer. Membranes were stripped using 0.2 M NaOH for consecutive detection of various proteins. The anti-ActB (I-19) and anti-Tsg101 (C-2) antibodies from Santa Cruz Biotechnology were used at a 1:1,000 dilution. Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology and used at a 1:1,000 dilution.
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RESULTS |
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A complete knockout of Tsg101 is deleterious at a very early stage during embryonic development. To test the hypothesis that Tsg101 is indispensable for embryonic development (32), we generated mice with a complete null mutation of Tsg101 by converting the floxed gene into a complete null allele in the female germ line of MMTV-Cre Tsg101fl/+ mice. Line A, which is one of several MMTV-Cre transgenic strains described previously (33, 34), exhibits Cre expression in developing oocytes. This feature makes this particular line useful to generate a complete null allele from any floxed locus. MMTV-Cre (line A) Tsg101fl/+ females were mated with wild-type males to generate Tsg101 heterozygous null mice (Tsg101+/-) and to segregate out the MMTV-Cre transgene. Tsg101+/- mice of both genders were selected from the resulting offspring. These heterozygous knockouts were maintained as a separate population and crossed among each other to generate Tsg101 homozygous null mutants (Tsg101-/-). Initially, 50 mice from six independent litters of heterozygous knockout intercrosses were genotyped. We were unable to identify a single Tsg101 knockout mouse. Surviving pups were either Tsg101+/+ (18 of 50) or Tsg101+/- (32 of 50). These results represented the Mendelian ratio of mutant and wild-type alleles when Tsg101 deficiency results in embryonic death. This observation further suggested that the wild-type Tsg101 locus was not imprinted.
Determination of the time point of embryonic death is important for the design of rescue experiments where viable tissues from a knockout can be transplanted into wild-type recipients to study the loss of function of important tumor suppressor genes in adult animals. To answer this question, we isolated and genotyped embryos at various time points (days E3.5, E6.5, and E12.5) from Tsg101 heterozygous intercrosses. We were able to identify Tsg101-/- blastocysts on day E3.5 which were indistinguishable in their overall morphology from wild-type littermates (Fig. 2A). However, we failed to clearly identify Tsg101 homozygous mutants after implantation. We isolated deciduae on day E6.5, and all embryos, even those few with an abnormal appearance (Fig. 2B, arrow), contained embryos that were identified as wild type or heterozygous mutants. Therefore, death in our animal model occurs at an earlier time point, most probably around implantation or very shortly thereafter. In summary, this experiment confirmed our hypothesis that Tsg101 is crucial for early embryonic development. Since the generation of Tsg101-deficient ES cells and the cultivation of the inner cell mass (ICM) of Tsg101 knockout blastocysts were unsuccessful (see Discussion), it is virtually impossible to rescue viable tissues from conventional Tsg101 knockout embryos and to generate somatic mutants through chimera production or transplantation of cells into adult recipients.
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Inactivation of both Tsg101 alleles in differentiating mammary epithelial cells results in impaired mammogenesis. To address whether the loss of Tsg101 function plays a role in normal mammogenesis and the development of mammary tumors, we generated mice that carry a WAP-Cre transgene in addition to two floxed alleles of the Tsg101 locus (WAP-Cre Tsg101fl/fl). The WAP-Cre transgene is specifically active in differentiating alveolar cells during late pregnancy and lactation. However, a significant number of recombined cells bypass apoptotic signals, survive remodeling during involution, and serve as progenitor cells for the alveolar compartment in subsequent pregnancies (30, 34). This feature makes the WAP-Cre strain an important tool to study tumor suppressor function in the mammary gland (39).
A WAP-Cre-mediated knockout of both alleles of Tsg101 leads to a severe inhibition of normal mammary gland development and the inability of mutant mice to support a litter (Fig. 3). Of 20 conditional knockout mice, 12 (60%) were completely unable to lactate after their first pregnancy, resulting in the loss of their entire litter within 24 h (Fig. 3A). On average, the few lactating conditional knockout mice supported only four or five pups (x = 4.5) by day 12 of lactation. The offspring were often malnourished. A histological examination of mammary tissues revealed that alveolar development was impaired in the Tsg101 conditional knockout mice (WAP-Cre Tsg101fl/fl) compared to their controls (Tsg101fl/fl) (Fig. 3B). It is a common feature of some genetically engineered mouse strains that lactation can be restored in successive pregnancies. To address this issue, we monitored lactation in conditional knockout dams and their controls for three consecutive gestation cycles (Fig. 3A). Interestingly, while some animals were able to establish lactation, almost half of all animals were still unable to nurse their young after the second and third pregnancies. Also, the average litter size that a lactating conditional knockout dam was able to support did not increase (x = 4.5 and 4.0, respectively). Therefore, a complete reversal of the phenotypic abnormalities of the mutant mice was not achieved. This suggested that either normal mammary epithelial cells have only a very limited capacity to bypass Tsg101 gene function or there is selective amplification of alveolar cells that maintain unrecombined alleles of Tsg101 due to WAP-Cre transgene silencing.
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Tsg101 deficiency results in the death of mammary epithelial cells. Since Tsg101 is expressed in all cell types of the mammary gland and throughout mammogenesis, we did not reason that Tsg101 is specifically required for terminal differentiation of the alveolar compartment. In addition, Tsg101 is preferentially deleted from late-differentiating cells expressing the Wap gene (i.e., WAP-Cre-mediated recombination). Therefore, it was not surprising that we observed only slightly lower levels of ß-casein and Wap mRNA in the conditional knockouts (data not shown). These marginal differences were probably caused by a smaller number of alveolar cells in the mutant mice than in their littermate controls (Fig. 3B). We postulated that Tsg101 could be important for cell growth and proliferation and that it may be crucial for cell survival since differentiating alveolar cells exhibit a much lower proliferation rate (35). This hypothesis is supported by the fact that there is a substantial negative selection against Tsg101-deficient cells during lactation (i.e., after completion of terminal differentiation). We used BrdU labeling and immunohistochemistry with the M30 CytoDEATH antibody to address whether impaired alveolar development is caused by a block in proliferation, an increase in cell death, or both in the WAP-Cre mediated conditional knockout of Tsg101 (Fig. 5). In particular, we studied the presence of dividing and apoptotic cells shortly after WAP-Cre becomes highly active (days 16 and 17 of gestation) and immediately postpartum, when milk secretion is initiated. Similar numbers of BrdU-labeled cells were observed in the knockouts and the controls at both stages of mammogenesis, suggesting that cell proliferation was not drastically reduced in the mutant mice (Fig. 5A to D). In general, apoptotic epithelial cells were barely visible in the mammary glands of wild-type mice at the onset of lactation, as determined by immunohistochemistry using the M30 CytoDEATH antibody (Fig. 5E). This antibody specifically recognizes a caspase-cleaved epitope of the keratin 18 (K18) protein (4). The cytoDEATH antibody recognizes only dying epithelial cells since K18 is a specific marker for the epithelial compartment of the mammary gland. In contrast to the controls, Tsg101 conditional knockout mice exhibited numerous apoptotic epithelial cells around parturition. The number of dying cells within a developing alveolus seemed to be larger in less well-organized lobular structures (compare Fig. 5F, G, and H). The staining intensity and number of dying cells were, however, irregular throughout entire cross sections, which made it virtually impossible to objectively quantify the exact percentage of apoptotic cells in vivo. Therefore, we reexamined cell growth and cell death in primary mammary epithelial cell cultures, where Cre-expressing cells can be selected and the deletion of Tsg101 can be controlled in a timely manner.
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DISCUSSION |
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exon 1-/- model, deficiency of this gene resulted in embryonic death around the time of implantation. This assumption was also verified recently in a different mouse model carrying a deletion of exons 8 and 9, which encode the coiled-coil domain near the C-terminal end of Tsg101 (24). These mice die between E5.5 and E6.5 due to a defect in cell proliferation and mesoderm formation. An increase in cell death was not observed in these mutants. In the Tsg101 exon 1-/- model, we were unable to identify homozygous mutant embryos at day E6.5. This suggests that there might be marginal differences in the phenotypes of the two mouse models depending on which functional domains were deleted from the Tsg101 locus. Since neither the generation of Tsg101-deficient ES cells nor the cultivation of the ICM from Tsg101 exon 8/9-/- blastocycts was successful (24), it is virtually impossible to rescue Tsg101-deficient cells by transplantation. To investigate the biological effects of Tsg101 deficiency on the proliferation and differentiation of breast epithelial cells and to verify the proposed function of this gene as a tumor suppressor for sporadic forms of breast cancer, we developed a knockout mouse with a mammary cell-specific deletion of Tsg101. Deficiency of Tsg101 in differentiating mammary epithelial cells resulted in impaired mammary development. One consequence was the inability of the mutant mice to lactate normally. This phenotype was caused mainly by apoptosis of differentiated cells, which normally exhibit a low proliferation rate (35). This phenomenon seems not to be a unique feature of mammary epithelial cells or of differentiating cells in general. We observed cell death as a consequence of Tsg101 deficiency in a variety of proliferating cell types including MEFs (11). In agreement with our findings, Garrus et al. (6) reported a slight growth inhibition of 293 cells when Tsg101 was transiently knocked down for 72 h by using the small interfering RNA approach. In addition, the introduction of Tsg101 siRNAs into human breast cancer cells (MCF-7) was highly toxic when these cells were treated repeatedly over time. Whereas Tsg101-deficient MCF-7 cells died, transfection of other siRNA control vectors had little or no effect on the growth of these cells (X. Lin and W. Nelson, John-Hopkins University, personal communication). In summary, these observations suggest that Tsg101 is important for the survival of nonimmortalized (e.g., mammary epithelial cells and MEFs) as well as immortalized and transformed (e.g., 293T and MCF-7 cells) cell lines. Our further studies of transformed Tsg101fl/fl MEFs, which were able to form tumors in nude mice, seemed to support this assumption. The Cre-mediated deletion of Tsg101 in explanted tumor cells resulted in instant cell death (M. D. Henry, A. Krempler, and K.-U. Wagner, unpublished data).
Tsg101 and cell cycle regulation. Our studies of Tsg101-deficient primary cells revealed that in addition to cell survival, Tsg101 is important for cell proliferation. The retroviral Cre-mediated conditional knockout of Tsg101 resulted in cell cycle arrest and death before the cells entered the S phase. These observations were consistent with our findings about the role of Tsg101 in proliferating embryonic fibroblasts (11). Tsg101-deficient MEFs did not incorporate BrdU and lacked expression of cyclins A and B. In addition, their cyclin E/Cdk2 complex was inactive, which clearly suggested a sustained G1/S arrest before Tsg101-deficient cells died. The mechanism responsible for the inactivation of Cdk2 in the conditional knockout cells needs to be identified. In a recent report, Oh et al. (22) showed that exogenous levels of Tsg101 in differentiating keratinocytes can positively regulate p21WAF-1/C1P-1, a suggested tumor suppressor protein that mediates G1 arrest in a p53-dependent manner (2, 5, 19). In proliferating keratinocytes, which exhibit stable levels of p21, overexpression of Tsg101 exerts growth suppression through modulation of p21 binding to the cyclin E/Cdk2 complex. It is possible that the mechanisms, which mediate growth arrest in Tsg101-deficient cells and the Tsg101 overexpression model, are the same. However, we did not observe a significant change in p27Kip1 and p21WAF-1/CIP-1 protein levels in the conditional knockouts, suggesting that Tsg101 is not essential for the stability of these major Cdk2 inhibitors in MEFs (11). Preliminary studies of Tsg101-/- MEFs showed a larger amount of phosphorylated Cdk2 (A. Krempler and K.-U. Wagner, unpublished data), and we are currently investigating whether inhibiting phosphorylations mediate inactivation of the cyclin E/Cdk2 complex in Tsg101-deficient cells.
The role of Tsg101 as a positive regulator of p21 and/or Cdk2 activity would provide a mechanism for the suggested function of this protein as an inhibitor of the cell cycle and a potential tumor suppressor. In contrast, Li et al. (15) reported a role for Tsg101 as a positive regulator of Mdm2, which is an oncogene that mediates the degradation of p53. The same group of investigators was able to verify their findings by using a Tsg101-deficient mouse model, which exhibited a cell cycle arrest mediated by upregulation of p53 and p21 (24). Through introduction of a p53-null mutation into the Tsg101 exon 8/9-/- background, Ruland et al. (24) were able to prolong the survival of mutant embryos for about 2 to 3 days. Given the suggested importance of Tsg101 as a key regulator of Mdm2, this rescue seemed to be marginal compared to that in Mdm2/p53 double-mutant mice, where a knockout of p53 completely rescued embryonic death caused by Mdm2 deficiency (9, 21). In addition, the suggested function for Tsg101 as a stabilizer of Mdm2 (i.e., an oncogene) is inconsistent with various reports that Tsg101 overexpression leads to cell cycle arrest and cell death (22, 32, 37, 40). The findings that Tsg101 deficiency resulted in cell cycle arrest through upregulation of p53 and p21 therefore contradict the mechanism proposed by Oh et al. (22), in which Tsg101 is a crucial mediator of p21 protein stability and of p21 function as a cell cycle inhibitor on the cyclin E/Cdk2 complex (see above). Our conditional knockout model did not reveal any involvement of p53 and its main effectors in the deleterious phenotype caused by Tsg101 deficiency (11). Various upstream (p19ARF, p16Ink4a, and Mdm2) and downstream (p21WAF-1/CIP-1 and p27Kip1) targets of p53 exhibited no significant change in their protein levels in Tsg101-/- MEFs. Furthermore, neither the functional inhibition of p53 nor the deletion of the p53 gene had a noticeable effect on the deleterious phenotype. Therefore, our findings do not support a biologically relevant function of Tsg101 in regulating the Mdm2-p53 feedback loop, as previously suggested (15, 24). On the other hand, Tsg101 deficiency causes a cell cycle arrest in addition to cell death. While p53 is not essential for mediating cell death in Tsg101-/- MEFs (11), more detailed analysis of whether p53 plays a role in cell cycle arrest observed in the Tsg101 conditional knockout model is needed.
Tsg101 and neoplastic transformation. Li and Cohen (13) reported that a functional knockout of Tsg101 using a conventional antisense approach resulted in instant neoplastic transformation of immortalized fibroblasts. However, the role of the Tsg101 gene as a tumor suppressor in human malignancies is still controversial. Genomic deletions and aberrant splice variants have been implicated in sporadic forms of breast cancer and a variety of other human malignancies (see, for example, references 7, 12, 14, 28, and 29). However, there are also numerous contradictory reports (see, for example, references 3, 17, 27, and 38). To verify whether the loss of function of Tsg101 is involved in neoplastic transformation in vivo, we maintained a large cohort of Tsg101 mutant mice (heterozygous complete and tissue-specific knockouts) for 2 years. These mice were closely monitored for tumor formation. Neither haploinsufficiency of Tsg101 (see also the report by Ruland et al. [24]) nor the deletion of both Tsg101 alleles in mammary epithelial cells resulted in tumor formation. Our data suggest that a loss of function of Tsg101 is insufficient to trigger neoplastic transformation; therefore, our observations contradict earlier reports by Li et al. (13, 14). Generally, in vitro models such as NIH 3T3 cells, which were used to identify Tsg101 and its tumor suppressive properties, are less reliable since these cells are immortal and carry numerous mutations in other tumor suppressor loci such as p53, p19Arf, or p16Ink4a (25). Specifically, the original NIH 3T3 cell line and its tumorigenic derivative (SL6 cells) described by Li and Cohen (13) are deficient in p19Arf and p16Ink4a (Krempler and Wagner, unpublished). More importantly, we showed recently that the antisense strategy used by Li et al. did not completely inhibit gene expression. SL6 cells still express a significant amount of the Tsg101 protein (11), and therefore it needs to be established (i) whether a knockdown triggers a different phenotype from a null mutation and (ii) whether antisense constructs target other genes with partial sequence similarity to Tsg101. In summary, the results of our studies on Tsg101 conditional knockout mice demonstrate that it is very important to generate genetically engineered mouse models with defined targeted mutations to verify a proposed tumor-suppressive function of genes identified in random mutagenesis screens in immortalized cell lines.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grant CA-93797 from the National Cancer Institute to K.U.W. The embryonic stem cell work was funded in part by the NIH Breast Cancer Think Tank. A.K. receives a stipend from the Deutsche Forschungsgemeinschaft (DFG, KR 2107/1-1).
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FOOTNOTES |
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Present address: Animal Science Research Center, University of Missouri, Columbia, MO 65211.
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