The DREAM complex is an important regulator of mitotic gene expression during the cell cycle. Here we report that inactivation of LIN9, a subunit of DREAM, results in premature senescence, which can be overcome by the SV40 large T (LT) antigen. Together with the observation that p16INK4a and p21Waf1 are upregulated upon loss of LIN9, these results indicate that senescence is triggered by the pRB and p53 tumor suppressor pathways. We also find that LIN9-null cells that escape senescence are chromosomally instable because of compromised mitotic fidelity. SV40 LT-expressing cells that adapt to the loss of LIN9 can grow anchorage-independently in soft agar, a hallmark of oncogenic transformation. Taken together, these results suggest an important role of mitotic gene regulation in the maintenance of genomic stability and tumor suppression.
Precise regulation of mitosis is important to prevent aneuploidy, a hallmark of most cancers (Holland and Cleveland, 2009). Aneuploidy can be caused by errors in mitosis and cytokinesis. For example, chromosome segregation errors due to defects in the mitotic spindle checkpoint can result in genomic instability and aneuploidy (Musacchio and Salmon, 2007). The spindle checkpoint delays anaphase until all chromosomes are aligned at the metaphase plate and attached to the spindle microtubule. Although the spindle checkpoint is essential for normal chromosome segregation and genomic stability, genes encoding for mitotic proteins are rarely mutated in tumors. This suggests that changes in the expression of spindle checkpoint proteins could have a role in genomic instability in cancer (Cimini and Degrassi, 2005).
The DREAM (or LINC) complex is a master regulator of mitotic gene expression during the cell cycle (Schmit et al., 2007, reviewed in Blais and Dynlacht, 2007; Litovchick et al., 2007; Osterloh et al., 2007; Pilkinton et al., 2007a; Knight et al., 2009). Several proteins that are involved in chromosome segregation and cytokinesis, such as Bub1 and survivin, are direct targets of DREAM (Osterloh et al., 2007; Schmit et al., 2007; Pilkinton et al., 2007b; Knight et al., 2009). DREAM consists of a five-protein core module and associated proteins (Litovchick et al., 2007; Schmit et al., 2007; Pilkinton et al., 2007a; Knight et al., 2009). In quiescent cells DREAM binds to the E2F4 transcription factor and to p130, which is related to the retinoblastoma tumor suppressor protein. In the S-phase, binding to E2F4/p130 is lost and another transcription factor, B-MYB, is incorporated into the complex. DREAM, together with E2F4 and p130, contributes to the repression of E2F-regulated genes in G0/G1, whereas DREAM–B-MYB is essential for gene activation in G2/M (Litovchick et al., 2007; Osterloh et al., 2007; Schmit et al., 2007; Pilkinton et al., 2007a; Knight et al., 2009). RNA interference-mediated knockdown of DREAM subunits in human cells inhibits their proliferation owing to inhibition of mitotic gene expression (Osterloh et al., 2007; Schmit et al., 2007).
To investigate the in vivo function of DREAM, we recently generated a conditional knockout mouse model for LIN9, a conserved core subunit of DREAM (Reichert et al., 2010). We found that loss of LIN9 leads to embryonic lethality at the peri-implantation stage. LIN9 is also essential for survival of adult mice. Deletion of LIN9 in adult mice results in rapid atrophy of the epithelium in the small intestine because of reduced proliferation. In mouse embryonic fibroblasts, loss of LIN9 resulted in severe defects in progression through mitosis and cytokinesis because of its essential role in mitotic gene expression (Reichert et al., 2010).
In this study, we determined the long-term consequences of DREAM inhibition. We found that loss of DREAM results in premature senescence of human and mouse fibroblasts. The SV40 large T (LT) antigen can overcome this senescence, indicating that senescence is triggered by the pRB and p53 tumor suppressor pathways. However, the mitotic fidelity of cells lacking LIN9 is compromised, and LIN9-null cells that escape senescence are chromosomally instable. These cells can also grow anchorage-independently in soft agar. Our observations indicate an important role of the DREAM complex in the maintenance of genomic stability.
Depletion of DREAM results in senescence of human fibroblasts
DREAM has roles in gene expression during the cell cycle, and is required for proliferation of human and mouse cells. To investigate the long-term consequence of DREAM inhibition, we depleted LIN9, a core subunit of the DREAM complex, by RNAi. Human BJ fibroblasts were infected with a recombinant retrovirus encoding a small hairpin RNA directed against LIN9. After 10–13 days in culture, LIN9-depleted cells exhibited a large and flat morphology, a phenotype that is commonly observed in senescent cells. Indeed, staining for senescence-associated β-galactosidase activity, a marker of senescent cells, confirmed senescence after loss of LIN9 (Figure 1a). As expected, control-infected BJ cells never became senescent and continued to proliferate. Immunoblot analysis showed induction of the cell cycle inhibitors p16INK4a and p21Waf1 in LIN9-depleted cells (Figure 1b). RNAi-mediated depletion of two further subunits of DREAM, LIN54 and B-MYB, also resulted in senescence of BJ cells, indicating that this phenotype is not an isolated function of LIN9 (Figure 1c).
Deletion of Lin9 results in senescence of primary MEFs
We next investigated primary mouse embryonic fibroblasts (MEFs) carrying a conditional floxed allele of Lin9 and CreERT2 recombinase (Lin9fl/flCreERT2). Addition of 4-hydroxytamoxifen (4-OHT) to the culture medium results in activation of the hormone-inducible CreER enzyme and in deletion of Lin9. Loss of LIN9 resulted in an increase in binuclear cells because of inhibition of cytokinesis and in inhibition of growth, as reported by us previously (Reichert et al., 2010 and data not shown). Two weeks after deletion of LIN9, MEFs became senescent, as previously described (Figure 2a; Reichert et al., 2010). Similar to what was observed in human cells, loss of LIN9 in two different MEF clones was also accompanied by induction of p16INK4a and p21Waf1 (Figure 2b). This suggests that senescence is mediated by the p16INK4a-pRB and/or p53-p21Waf1 tumor suppressor pathways.
SV40 LT antigen overcomes the senescence of LIN9-null MEFs
To analyze whether senescence upon loss of LIN9 is dependent on pRB and/or p53, we used the SV40 LT antigen, which binds to and disrupts the function of the p53 and pRB tumor suppressors. We also used two mutants of LT, LT-K1 and LT-Δ434-444, to test which pathway is involved in senescence. The LT-K1 mutant can no longer bind to and inactivate pRB and thus only targets p53 (Zalvide and DeCaprio, 1995). LT-Δ434-444 cannot bind to p53 and only targets pRB and the related ‘pocket proteins’ p107 and p130 (Kierstead and Tevethia, 1993). Early-passage primary Lin9fl/flCreERT2 MEFs were infected with retroviruses encoding for SV40 LT wild type or mutants and were selected for antibiotic resistance (Figure 3a). Expression of LT wild type and mutants was verified by western blotting (Figure 3b). Expression of all three LT antigens was detectable, although expression of LT Δ434-444 was much weaker compared with LT wild type and LT K1, as has been observed previously (Ye et al., 2007). To confirm that the retinoblastoma pathway has been targeted efficiently by the Δ434-444 mutant, we analyzed levels of p130. It has been shown that SV40 LT species that bind to the pocket proteins alter the phosphorylation state and steady-state levels of p130 (Stubdal et al., 1996, 1997; Chao et al., 2000). We found low levels of p130 in cells expressing wild-type LT and Δ434-444, and high levels of p130 in cells expressing the LT K1 mutant. This clearly indicates that the retinoblastoma pathway has been targeted efficiently in cells expressing LT wild type and Δ434-444.
Deletion of Lin9 was induced in the different cultures by addition of 4-OHT (Figure 3a). Two weeks later, senescence was scored by senescence-associated β-galactosidase staining. Wild-type LT was able to block the senescence phenotype after deletion of Lin9 (Figure 3d). Interestingly, however, neither LT K1 nor LT Δ434-444 were able to prevent the senescence phenotype (Figure 3d). Thus, although the combined inactivation of pRB and p53 prevented senescence after loss of LIN9, inactivation of either p53 or pRB alone was insufficient to prevent senescence. We conclude that both the pRB and p53 pathways can independently mediate senescence upon loss of LIN9.
Cells can adapt to the loss of LIN9
Next, we asked whether SV40 LT not only prevents senescence but can also rescue the growth inhibition and errors in mitosis associated with the loss of LIN9. To address this question, Lin9 was deleted from Lin9fl/flCreERT2 (LT) MEFs by the addition of 4-OHT. Further, proliferation and the fraction of binuclear cells were determined and compared with control-treated cells. As shown in Figure 4a, proliferation of SV40 LT-expressing cells was strongly reduced, but not completely inhibited after deletion of Lin9. Importantly, 4-OHT had no effect on proliferation of SV40 LT-expressing Lin9+/+CreERT2 MEFs, indicating that inhibition of proliferation is not a toxic effect of 4-OHT or of activated Cre-recombinase. After 4-OHT treatment the fraction of binuclear cells increased from 5 to over 25% in LT Lin9fl/flCreERT2 MEFs, similar to what was observed in primary Lin9fl/flCreERT2 MEFs (Figure 4b). Thus, although SV40 LT is able to block senescence after deletion of LIN9 (Figure 3), it is insufficient to overcome the cytokinesis defect and prevent formation of binuclear cells. In addition, SV40 LT did not completely overcome the growth inhibition associated with the loss of LIN9.
To better assess the number of LT-expressing Lin9fl/flCreERT2 cells that were able to proliferate in the absence of LIN9, we performed colony-forming assays. 4-OHT-treated and untreated Lin9fl/flCreERT2 (LT) MEFs were plated at low density and the number of colonies was determined after 14 days. Consistent with the reduced growth observed above, colony formation was significantly reduced, but not completely inhibited after 4-OHT treatment (Figure 4c). Importantly, PCR genotyping confirmed deletion of Lin9 in 4-OHT-treated cultures after 3 weeks, indicating that colonies in 4-OHT-treated cultures do not arise because of inefficient deletion of the Lin9 allele (Figure 4d). We concluded that LT-immortalized MEFs can adapt to the loss of LIN9.
Cells that adapt to the loss of LIN9 are aneuploid
To characterize in more detail how cells adapt to the loss of LIN9, single colonies from 4-OHT-treated Lin9fl/flCreERT2 MEFs expressing SV40 LT were isolated. Some, but not all, clones of these single clones could be expanded in culture. We also isolated and expanded control clones from untreated Lin9fl/flCreERT2 (LT) MEFs.
To characterize these clones, we performed karyotype analyses from metaphase spreads generated from three control clones and three Lin9-null clones (Figure 5). Two control clones generated from wild-type cells contained a mixture of near-diploid and near-tetraploid cells and showed little aneuploidy (Figures 5a and b). The third control clone had a near-tetraploid karyotype, with more than 94% of the cells containing 71–80 chromosomes. In contrast, all three Lin9-mutant clones contained no diploid cells. Instead, in all three Lin9-deficient clones high percentages of cells with abnormal karyotypes were detected (Figures 5a and b). Overall, 10–20% of the cells of Lin9 mutant clones contained over 101 chromosomes, indicating that Lin9-depleted cells become highly aneuploid.
Cells that adapt to the loss of LIN9 grow independently of anchorage
Because of the high aneuploidy in Lin9-null cells, and because aneuploidy is a characteristic feature of tumor cells, we next scored the oncogenic transformation of Lin9-deficient clones. To do so, the ability of clones to grow independently of anchorage in soft agar was determined. Control clones only formed very few and small clones in soft agar, as expected (Figure 6). In striking contrast, all three Lin9-deficient clones formed large and fast-growing colonies in soft agar, indicating that they are transformed (Figure 6).
Mitotic gene expression is independent of LIN9 in cells that have adapted to the loss of Lin9
Next, we wanted to understand how cells adapt to the loss of LIN9. LIN9 is a core subunit of the DREAM complex, which functions as a master activator of mitotic genes during the cell cycle. Deletion of Lin9 in primary cells results in downregulation of a cluster of mitotic genes, and consequently in growth arrest and senescence (Reichert et al., 2010). It is possible that expression of mitotic genes becomes independent of DREAM in cells that express the LT antigen. To address this possibility, we first analyzed gene expression in pools of Lin9fl/flCreERT2 (LT) MEFs, directly after infection with retrovirus encoding the LT antigen. RNA was isolated before and after addition of 4-OHT. Expression of selected mitotic genes Nusap1, Aspm and Cenp-f, which are required for normal mitotic progression, was analyzed by reverse transcriptase–qPCR. We also analyzed the expression of Gas2l3, a novel mitotic DREAM target gene (unpublished observation). As can be seen in Figure 7, expression of mitotic DREAM target genes was reduced by approximately 50–60% after loss of LIN9. As a control, we analyzed the expression of the G1/S-specific B-MYB mRNA, which was unchanged upon 4-OHT treatment. These results indicate that acute expression of the SV40 LT antigen is not sufficient to overcome the requirement for LIN9 in mitotic gene expression, consistent with the finding that LIN9 is required for proliferation and cytokinesis in LT-immortalized MEFs (Figures 4a and b).
Next, we compared mitotic gene expression in three individual clones established from 4-OHT-treated cells and in a control clone (Figure 7). Interestingly, expression of mitotic genes was not significantly lower in LIN9-deficient clones as compared with the control clone. This indicates that cells that have adapted to the loss of LIN9 no longer depend on LIN9 for expression of mitotic genes.
In recent studies, the DREAM complex has emerged as a master regulator of mitotic gene expression during the cell cycle (Litovchick et al., 2007; Osterloh et al., 2007; Schmit et al., 2007; Pilkinton et al., 2007a; Knight et al., 2009). Loss of LIN9, a core subunit of DREAM, in conditional knockout MEFs severely abolishes the ability of the cells to proliferate and to progress through mitosis and cytokinesis because of reduced expression of mitotic genes (Reichert et al., 2010). In this study, we investigated the long-term consequence of loss of LIN9 in MEFs and found that deletion of LIN9 induces cellular senescence. A similar phenotype was also observed after small hairpin RNA-mediated depletion of DREAM subunits in human BJ fibroblasts. Thus, a consequence of impaired DREAM function is the induction of a permanent cell cycle arrest. Senescence is considered an important fail-safe mechanism to prevent tumorigenesis. It is induced in response to stress such as DNA damage, oncogene activation and oxidative stress (Adams, 2009). Errors in progression through mitosis, caused for example by defects in the spindle checkpoint, can also induce senescence. For example, MEFs with reduced levels of the spindle checkpoint proteins BubR1 and Bub1 show premature senescence (Baker et al., 2004; Schliekelman et al., 2009). Similarly, centromere dysfunction due to a reduction in the kinetochore protein CENP-A can induce senescence of human fibroblasts (Maehara et al., 2010). As DREAM is required for expression of many proteins that are essential for mitosis, including CENP-A and Bub1, it is reasonable to suggest that senescence upon the loss of LIN9 is caused by errors in mitosis and cytokinesis because of reduced expression of proteins involved in mitotic progression. Interestingly, a recent study demonstrated that LIN9 expression is downregulated in senescent cells (Song et al., 2010). As it is not clear whether this reduction is the cause or consequence of cellular senescence, further studies are necessary to investigate whether there is a positive-feedback loop between DREAM inhibition and the cellular response to stress.
The upregulation of p16INK4a and p21Waf1 in LIN9-deficient cells and the fact that senescence of LIN9-deficient cells can be overcome by expression of the SV40 LT antigen indicated that premature senescence of these cells is dependent on the p53 and pRB tumor suppressor pathways. Although the exact pathways that result in upregulation of p16INK4a and p21Waf1 have to be investigated, it appears that senescence is an important fail-safe mechanism that prevents cells from undergoing defective mitoses. We found that once senescence is overcome by the inactivation of p53 and pRB, the mitotic fidelity of LIN9-null cells is compromised. This results in propagation of cells with chromosomal abnormalities, as indicated by high levels of aneuploidy in cells that have lost LIN9. This chromosomal instability of Lin9-deleted cells leads to anchorage-independent growth, a defining feature of oncogenically transformed cells. Thus, the induction of senescence in response to abnormal progression through mitosis appears to suppress the production of potentially harmful cells.
It is surprising that, once senescence is overcome, cells can tolerate the loss of LIN9, despite its essential function in embryonic development, and in proliferation in adult mice and MEFs (Reichert et al., 2010). Interestingly, cells that have adapted to the loss of LIN9 no longer depend on LIN9 for their mitotic gene expression. It is possible that other transcription factors such as FoxM1, which are also important for expression of G2/M genes, compensate for the loss for LIN9 in these cells (Laoukili et al., 2005). How cells become independent of LIN9 is currently being investigated.
Taken together, in this study we have demonstrated that altered mitotic gene expression results in genomic instability and aneuploidy and that this is linked to malignant transformation. Our observations contribute to the understanding of the mechanisms that underlie the development of aneuploidy in cancer cells.
Materials and methods
Mouse embryonic fibroblasts
Primary MEFs were isolated from 13.5 dpc Lin9fl/fCreERT2 embryos as previously described (Gaubatz et al., 2000). MEFs were cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Invitrogen, Darmstadt, Germany). Deletion of Lin9 was achieved by treatment with 1 μM 4-OHT for 48 h. For growth curves, MEFs were plated in triplicate in 24-well plates. At the indicated time points, cells were fixed in 10% formalin and stained with crystal violet. The dye was extracted and the optical density determined as described previously (Serrano, 1997).
Expression of SV40 LT antigen
Early-passage (p2) Lin9fl/fCreERT2 MEFs were infected with retroviral expression constructs encoding SV40 LT, LT K1 or LTΔ434-44 as described (Cotsiki et al., 2004; Gagrica et al., 2004). Cells were selected with 400 μg/ml neomycin for 5 days.
Colony formation assay
Lin9fl/fCreERT2 MEFs treated with 4-OHT or solvent were seeded at a density of 3200 cells per 10-cm dish and cultured for 14 days. Cells were fixed and stained with crystal violet.
1 × 105 cells were transferred to 2 ml Dulbecco’s modified Eagle’s medium containing 0.35% low-gelling agarose and seeded in triplicate into six-well plates containing a 2-ml layer of solidified 0.7% agarose in complete medium. After 10 days, the number of foci was scored.
Total RNA was isolated with Trizol (Invitrogen), reverse transcribed with 0.5 units of M-MLV-RT Transcriptase (Thermo Scientific, Bonn, Germany) and analyzed with quantitative real-time PCR with SYBR Green reagents from Thermo Scientific using the Mx3000 (Stratagene, Waldbronn, Germany) detection system. Expression differences were calculated as described before (Schmit et al., 2007). The following primers were used: Nusap1: fw 5′-TCTAAACTTGGGAACAATAAAAGGA-3′, bw 5′-TGGATTCCATTTTCTTAAAACGA-3′; Aspm: fw 5′-GATGGAGGCCGAGAGAGG-3′, bw 5′-CAGCTTCCACTTTGGATAAGTATTTC-3′; Cenpf: fw 5′-AGCAAGTCAAGCATTTGCAC-3′, bw 5′-GCTGCTTCACTGATGTGACC-3′; Hprt: fw 5′-TCCTCCTCAGACCGCTTTT-3′, bw 5′-CCTGGTTCATCATCGCTAATC-3′.
To prepare metaphase spreads, cells were incubated with 50 ng/ml colcemid for 6 h. Cells were collected by trypsinization, washed in PBS and suspended in 75 mM KCl. After incubation at room temperature for 15 min, cells were fixed in 75% methanol and 25% acetic acid. Aliquots were dropped onto slides and stained with Giemsa solution. Chromosome numbers were determined by light microscopy.
Cells were lysed in TNN (50 mM Tris pH 7.5, 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 10 mM Na4P2O7, 2 mM Na3VO4, 100 mM NaF, 1 mM Dithiothreitol, Protease Inhibitor Mix (Sigma, Munich, Germany)). Proteins were separated by sodium dodecyl sulfate (SDS) gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane and detected by immunoblotting with the following antibodies: p16 (H156 and F-12, Santa Cruz, Santa Cruz, CA, USA), p21 (EA10, Calbiochem, Darmstadt, Germany and C-19, Santa Cruz), tubulin (B-5-1, Sigma), actin (C4, Santa Cruz), SV40 LT (Pab108, Santa Cruz), p130 (C-20, Santa Cruz).
Senescence-associated β-galactosidase staining
Senescence-associated β-galactosidase activity was detected as described (Dimri et al., 1995).
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We thank all members of the laboratory for their suggestions and critical reading of the manuscript. We also thank Michael Schmid and Claus Steinlein for their advice and help with karyotype analysis. This work was supported by grants from the DFG (575/6-1 and TR17-B1) to SG.
The authors declare no conflict of interest.
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Cite this article
Hauser, S., Ulrich, T., Wurster, S. et al. Loss of LIN9, a member of the DREAM complex, cooperates with SV40 large T antigen to induce genomic instability and anchorage-independent growth. Oncogene 31, 1859–1868 (2012). https://doi.org/10.1038/onc.2011.364
- DREAM complex
- genomic instability
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