Single cell tracking reveals that Msh2 is a key component of an early-acting DNA damage-activated G2 checkpoint

Dysfunction of cell-cycle checkpoints in DNA mismatch repair (MMR)-deficient cells in response to DNA damage has implications for anticancer therapy and genetic instability. We have studied the cell-cycle effects of MMR deficiency (Msh2(-/-)) in primary mouse embryonic fibroblasts (MEFs) exposed to cisplatin (10 microM x 1 h) using time-lapse microscopy. Kinetic responses of MEFs from different embryos and passage ages varied, but we report a consistent drug-induced inhibition of mitotic entry (approx. 50%). There was a loss of an early-acting (<5 h) delay in G2 to M transition in Msh2(-/-) cells, although a later-acting G2 arrest was apparently normal. This suggests that Msh2 primarily acts to delay mitotic entry of cells already in G2, that is, DNA damage incurred during G2 does not influence the cell once committed to mitotic traverse. Irrespective of Msh2 status, cisplatin treatment and the incurred DNA damage did not effect mitotic traverse or show any evidence for early (within 24 h) cell death. The results indicate that Msh2(-/-) status can result in the premature commitment to mitosis of a cell subpopulation, determined by the fraction residing in G2 at the time of damage induction. The findings suggest a new route to MMR-driven genetic instability that does not rely primarily on the integrity of the late-acting checkpoint.     

Msh2 deficiency leads to chromosomal abnormalities, centrosome amplification, and telomere capping defect

Msh2 is a key mammalian DNA mismatch repair (MMR) gene and mutations or deficiencies in mammalian Msh2 gene result in microsatellite instability (MSI+) and the development of cancer. Here, we report that primary mouse embryonic fibroblasts (MEFs) deficient in the murine MMR gene Msh2 (Msh2(-/-)) showed a significant increase in chromosome aneuploidy, centrosome amplification, and defective mitotic spindle organization and unequal chromosome segregation. Although Msh2(-/-) mouse tissues or primary MEFs had no apparent change in telomerase activity, telomere length, or recombination at telomeres, Msh2(-/-) MEFs showed an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA. These data suggest that MSH2 helps to maintain genomic stability through the regulation of the centrosome and normal telomere capping in vivo and that defects in MMR can contribute to oncogenesis through multiple pathways.     

Activation of the p53-dependent G1 checkpoint response in mouse embryo fibroblasts depends on the specific DNA damage inducer

The p53 tumor suppressor protein inhibits proliferation by inducing either cell cycle arrest or apoptosis in response to cellular stresses. Mouse embryo fibroblasts (MEFs) provide a primary cell model system in which to examine both functions of p53. MEFs treated with gamma-rays undergo p53-dependent G1 arrest, while oncogene-expressing MEFs treated with a variety of DNA-damaging agents undergo p53-dependent apoptosis. Although the p53-dependent G1 arrest checkpoint response to gamma-rays in MEFs has been well characterized, the response to other DNA-damaging agents has not. Here, we examine the effects of commonly utilized chemotherapeutics, including doxorubicin, etoposide, and cisplatin, on cell cycle arrest in MEFs, and we define the p53 dependence of these effects. In addition, we examine the response of MEFs to ultraviolet light (UVC), as a representative agent acting by inducing pyrimidine dimers. Although p53 is clearly activated by all the agents examined, as measured by p21 induction, there are surprising differences in the activities of these agents. For example, doxorubicin but not cisplatin can effectively induce a p53-dependent G1 arrest. UVC, in contrast, induces a p53-independent G1 arrest response. Thus, the exact response of cells to DNA damage depends on the specific agent used.     

The ability to engage enterocyte apoptosis does not predict long-term crypt survival in p53 and Msh2 deficient mice

Apoptosis and long term enterocyte survival were examined in vivo after exposure to three cytotoxic agents (Cisplatin, Nitrogen Mustard and N-methyl-N-nitrosourea (NMNU/MNU)) within mice either singly or doubly mutant for p53 and Msh2. P53 deficiency caused abrogation of the immediate apoptotic response to each agent, but only led to increased survival after cisplatin treatment. Msh2 deficiency reduced the apoptotic response to each agent, but only led to increased crypt survival after NMNU treatment. Following cisplatin treatment, the response of (Msh2(-/-), p53(-/-)) mice paralleled that of the p53(-/-) mice. A delayed wave of apoptosis was observed in both p53(-/-) and (Msh2(-/-), p53(-/-)) mice demonstrating this phenomenon to be independent of functional Mismatch repair (MMR). We conclude that loss of either p53 or Msh2 dependent apoptosis does not predict long-term crypt survival in vivo, however genetic status clearly can modulate survival for some agents such as cisplatin.     

Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint

The S-phase DNA damage checkpoint is activated by DNA damage to delay DNA synthesis allowing time to resolve the replication block. We previously discovered the p53-dependent S-phase DNA damage checkpoint in mouse zygotes fertilized with irradiated sperm. Here, we report that the same p53 dependency holds in mouse embryonic fibroblasts (MEFs) at low doses of irradiation. DNA synthesis in p53 wild-type (WT) MEFs was suppressed in a biphasic manner in which a sharp decrease below 2.5 Gy was followed by a more moderate decrease up to 10 Gy. In contrast, p53-/- MEFs exhibited radioresistant DNA synthesis below 2.5 Gy whereas the cells retained the moderate suppression above 5 Gy. DNA fiber analysis revealed that 1 Gy irradiation suppressed replication fork progression in p53 WT MEFs, but not in p53-/- MEFs. Proliferating cell nuclear antigen (PCNA), clamp loader of DNA polymerase, was phosphorylated in WT MEFs after 1 Gy irradiation and redistributed to form foci in the nuclei. In contrast, PCNA was not phosphorylated and dissociated from chromatin in 1 Gy-irradiated p53-/- MEFs. These results demonstrate that the novel low-dose-specific p53-dependent S-phase DNA damage checkpoint is likely to regulate the replication fork movement through phosphorylation of PCNA.     

p53 protects from replication-associated DNA double-strand breaks in mammalian cells

Genetic instability caused by mutations in the p53 gene is generally thought to be due to a loss of the DNA damage response that controls checkpoint functions and apoptosis. Cells with mutant p53 exhibit high levels of homologous recombination (HR). This could be an indirect consequence of the loss of DNA damage response or p53 could have a direct role in HR. Here, we report that p53-/- mouse embryonic fibroblasts (MEFs) exhibit higher levels of the RAD51 protein and increased level of spontaneous RAD51 foci Agents that stall replication forks, for example, hydroxyurea (HU), potently induce HR repair and RAD51 foci. To test if the increase in RAD51 foci in p53-/- MEFs was due to an increased level of damage during replication, we measured the formation of DNA double-strand breaks (DSBs) in p53+/+ and p53-/- MEFs following treatments with HU. We found that HU induced DSBs only in p53-/- MEFs, indicating that p53 is involved in a pathway to protect stalled replication forks from being collapsed into a substrate for HR. Also, p53 is upregulated in response to agents that inhibit DNA replication, which supports our hypothesis. Finally, we observed that the DSBs produced in p53-/- MEFs did not result in a permanent arrest of replication and that they were repaired. Altogether, we suggest that the effect of p53 on HR and RAD51 levels and foci can be explained by the idea that p53 suppresses formation of recombinogenic lesions.     

Inhibition of v-Abl transformation by p53 and p19ARF

Tumorigenesis is a multistep process that involves the activation of oncogenes and the inactivation of tumor suppressor genes. The transforming activity of the v-Abl oncogene of Abelson murine leukemia virus (A-MuLV) in immortal cell lines has been well studied, while the effects of v-Abl in primary fibroblasts are less clear. Here we show that v-Abl causes cell cycle arrest in primary mouse embryonic fibroblasts (MEFs) and elevated levels of both p53 and the cyclin-dependent kinase inhibitor p21Cip. p53-/- or p19ARF-/- MEFs were resistant to v-Abl-induced cell cycle arrest. Although wild-type MEFs were resistant to v-Abl transforming activity, p53-/- or p19ARF-/- MEFs were susceptible. The results indicate that loss of p19ARF and p53 function plays an important role during the transformation of primary cells by v-Abl. We suggest that although v-Abl is a potent oncogene, its full potential transforming activity cannot be realized until the ARF-, and p53-dependent growth inhibitory pathway is disabled. We also show that p53 is not the mediator of v-Abl toxicity in immortal fibroblasts and does not determine the susceptibility of immortal fibroblasts to v-Abl transformation.     

Msh2 deficiency does not contribute to cisplatin resistance in mouse embryonic stem cells

Several reports have suggested that a defect in the DNA mismatch repair (MMR) system not only causes resistance to methylating agents but also confers low-level resistance to the chemotherapeutic drug cisplatin. Here we report that in a clonogenic assay, mouse embryonic stem (ES) cells deficient for the MMR protein MSH2 respond similarly as wild-type cells to cisplatin. Furthermore, restoring MSH2 expression in a cisplatin-resistant subclone selected from an Msh2(-/-) cell population did not sensitize cells to cisplatin. To ascertain that our observations were not the result of a mutation in the Msh2(-/-) cells that obscured the contribution of a defective MMR machinery to cisplatin resistance, we made use of the Cre-lox system to create a cell line in which the Msh2 gene can be conditionally inactivated. However, while de novo inactivation of Msh2 rendered cells tolerant to the methylating drug N-methyl-N'-nitro-N-nitrosoguanidine as expected, it did not alter the sensitivity to cisplatin. In addition, we were not able to derive cisplatin-resistant subclones from this freshly generated MMR-deficient cell line. Thus, in ES cells we did not find evidence for direct involvement of MMR deficiency in cisplatin resistance.     

Msh-2 suppresses in vivo mutation in a gene dose and lesion dependent manner.

Mice deficient for the mismatch repair (MMR) gene Msh2 show accelerated tumourigenesis and a reduced apoptotic response to DNA damage of methylation type. Here we examine the effect of mutation for Msh2 on in vivo mutation frequencies in the intestine as determined by loss of function at the Dolichos biflorus (Dlb-1) locus. Spontaneous mutation frequencies were scored in cohorts of ageing mice either wild type or mutant for Msh2. In mice less than 1 year old, mutation frequencies were only elevated in Msh2 null mice. However, beyond this age heterozygous Msh2 mice showed significantly higher mutation frequencies than controls. These findings implicate a gene dose dependent requirement for Msh2 in mutation suppression and prompted an analysis of young Msh2 mutants following exposure to DNA damage. Following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), Msh2 deficient mice show a reduced apoptotic response and an increase in mutation frequency. Heterozygotes did not differ from controls. Following exposure to cisplatin, no significant elevation was seen in mutation frequencies, even within homozygotes. This is particularly surprising given the association between cisplatin resistance and MMR deficiency. These findings therefore demonstrate a complex reliance upon functional Msh2 in mutation surveillance. We have identified three separate scenarios. First, where retention of both Msh2 alleles over an extended period of time appears critical to the suppression of spontaneous mutation; second, 3 weeks following exposure to MNNG, where only complete loss of Msh2 results in elevated mutation; and finally following cisplatin exposure, where induced levels of mutation are independent of Msh2 status.     

Spontaneous and mutagen-induced transformation of primary cultures of Msh2-/- p53-/- colonocytes

Loss of function of mismatch repair (MMR) genes underlies hereditary nonpolyposis colorectal cancer (HNPCC). However, the inability to maintain primary colon epithelial cells in culture has limited the analysis of the contribution of MMR gene defects to colorectal tumorigenesis. We have now established primary cultures of epithelial cells from the colon crypts of Msh2-/- p53-/- double-knockout mice. These cells undergo spontaneous transformation (soft agar colonies and s.c. tumor formation), with a progressively shorter latency as a function of increasing passages in culture. Treatment of early passage cells with the mutagen methylmethane thiosulfonate (MMS) further decreases the transformation latency of Msh2-/- p53-/- cells. Spontaneous transformation of p53-/- colonocytes is only observed using late passage cells, and methylmethane thiosulfonate-treated early passage p53-/- colonocytes do not form tumors when injected into immunodeficient mice. Together, these findings support the pathogenic role of MMR gene inactivation in colorectal tumorigenesis and provide an experimental model for the serial assessment of the molecular phenotype associated with Msh2 deficiency.     

p8-deficient fibroblasts grow more rapidly and are more resistant to adriamycin-induced apoptosis

p8 is a stress-induced DNA-binding protein, biochemically related to the architectural chromatin binding HMG protein family and whose function is presently unknown. We obtained fibroblast from mice lacking p8 and found that p8 is involved in cell growth regulation and in apoptosis. p8(-/-) mouse embryonic fibroblasts (MEFs) grow more rapidly than p8(+/+) MEFs. This might be explained by the higher intracellular level and activity of the Cdk2 and Cdk4 observed in p8(-/-) MEFs, which in turn may result, at least in part, from the concomitant decrease observed in the amount of cyclin-dependent kinase inhibitor p27. We also report that p8 mRNA expression is strongly activated in fibroblasts after cell growth arrest induced by serum deprivation or confluence. As expected, MEFs expressing p8 arrest their growth more rapidly after serum deprivation than MEFs lacking p8, which strongly suggests that p8 over-expression is implicated in cell growth arrest. On the other hand, p8(+/+) MEFs are more sensitive than p8(-/-) MEFs to the apoptosis induced by adriamycin treatment. p53 might be involved, as p8 expression increases its intracellular amount and trans-activation capacity. Finally, demonstration that p53 is a negative trans-activator of p8 suggests the presence of a complex autoregulatory loop. In conclusion, p8 is a cell growth inhibitor that facilitates apoptosis induced in fibroblasts by DNA damage.     

Hypersensitivity of Brca1-deficient MEF to the DNA interstrand crosslinking agent mitomycin C is associated with defect in homologous recombination repair and aberrant S-phase arrest

Hypersensitivity of Brca1-deficient cells to interstrand crosslinking (ICL) agents such as cisplatin and mitomycin C (MMC) implicates an important role for Brca1 in cellular response to the ICL DNA damage repair. However, the detailed mechanism of how Brca1 is involved in the ICL response remains unclear. In this study, we analysed the cellular response to MMC treatment using isogenic mouse embryonic fibroblasts (MEFs) including wild type, p53-/- and p53-/-Brca1-/-. Marked hypersensitivity of p53-/- Brca1-/- MEFs to MMC was found, and the reconstitution of Brca1 expression in these cells restored resistance to MMC. Upon MMC treatment, wild-type MEF was temporarily arrested at G2/M phase but subsequently resumed a normal cell cycle progression. In contrast, Brca1-deficient MEF exhibited a marked time-dependent accumulation of cells arrested at S phase and a prolonged increase in the G2/M population, followed by extensive cell deaths. Importantly, DNA damage-induced Rad51 foci were not formed in these cells, suggesting a defect in homologous recombination. Such defects are fully rescued by reconstitution of Brca1 expression in Brca1-deficient MEF, suggesting that Brca1 directly plays an essential role in ICL repair, which depends on homologous recombination during S phase.     

Mitogen requirement for cell cycle progression in the absence of pocket protein activity

Primary mouse embryonic fibroblasts lacking expression of all three retinoblastoma protein family members (TKO MEFs) have lost the G1 restriction point. However, in the absence of mitogens these cells become highly sensitive to apoptosis. Here, we show that TKO MEFs that survive serum depletion pass G1 but completely arrest in G2. p21CIP1 and p27KIP1 inhibit Cyclin A-Cdk2 activity and sequester Cyclin B1-Cdk1 in inactive complexes in the nucleus. This response is alleviated by mitogen restimulation or inactivation of p53. Thus, our results disclose a cell cycle arrest mechanism in G2 that restricts the proliferative capacity of mitogen-deprived cells that have lost the G1 restriction point. The involvement of p53 provides a rationale for the synergism between loss of Rb and p53 in tumorigenesis.     

The alternative reading frame tumor suppressor inhibits growth through p21-dependent and p21-independent pathways

The alternative reading frame (ARF) tumor suppressor mediates growth arrest or apoptosis through activation of the p53 tumor suppressor. A prevailing concept is that ARF uses p21Cip1/Waf1, a p53-responsive gene and cyclin-dependent kinase (Cdk) inhibitor, to block cell cycle progression. Using p21 nullizygous cells, we demonstrate that p21 is nonessential for the antiproliferative activity of ARF and p53, although it likely governs the arrest through Cdk inactivation when present. ARF overexpression in p21-positive and p21-negative mouse embryo fibroblasts (MEFs), but not in primary cells lacking p53, induced a biphasic (G1 and G2) cell cycle arrest. The ARF-induced growth arrest, regardless of p21 status, coincided with activation of p53 and accumulation of hypophosphorylated retinoblastoma protein (retinoblastoma protein). In ARF-arrested p21-positive cells, the presence of growth-inhibitory retinoblastoma protein correlated with an absence of Cdk2-dependent kinase activity, an increase in p21 association with inactive Cdks, and a lack of cyclin A expression. In contrast, p21-/- mouse embryo fibroblasts were arrested by ARF despite containing elevated levels of cyclin A protein and highly active Cdk2-dependent kinases. These findings provide evidence that ARF can block growth through a p21-independent pathway(s) that overrides Cdk2 activation.     

A combination of MSH2 DNA mismatch repair deficiency and expression of the SV40 large T antigen results in cisplatin resistance of mouse embryonic fibroblasts

Mutations in the human mismatch repair (MMR) genes are associated with hereditary non-polyposis colorectal cancer as well as other sporadic cancers. MMR gene mutations have been implicated in the resistance of human tumours to cisplatin and several tumour-derived MMR-deficient cells show cisplatin resistance in vitro. In addition, hypoxia, a common feature of the tumour microenvironment, has been shown to influence tumour responses to conventional cancer treatments. We have examined the role of the mMSH2 MMR protein on repair of cisplatin-damaged DNA and cisplatin sensitivity in mMSH2-deficient murine fibroblasts and mMSH2-proficient controls under conditions of normoxia and hypoxia. Sensitivity to cisplatin was measured using the MTT assay and clonogenic survival. Repair of cisplatin-damaged DNA was measured using a host cell reactivation (HCR) assay employing a non-replicating recombinant virus expressing the β-galactosidase reporter gene. Sensitivity to cisplatin was significantly less and HCR of the cisplatin-damaged reporter gene was significantly greater in SV40-transformed mMSH2-deficient cells (MS5-7) compared to mMSH2-proficient controls (BC1-6) under both normoxic and hypoxic conditions. In contrast, sensitivity to cisplatin was significantly greater and HCR was similar in primary mMSH2-deficient compared to mMSH2-proficient murine fibroblasts under both normoxic and hypoxic conditions. Sensitivity to cisplatin was also significantly greater and HCR was similar in primary mMSH2-deficient compared to mMSH2-proficient murine fibroblasts transfected with a control plasmid under both normoxic and hypoxic conditions. In contrast, sensitivity to cisplatin was less and HCR was similar in primary mMSH2-deficient compared to mMSH2-proficient murine fibroblasts transfected with a plasmid expressing SV40 large T antigen under both normoxic and hypoxic conditions. These results suggest that loss of MMR alone does not result in increased resistance to cisplatin in murine fibroblasts and that additional concomitant alterations in cells expressing the SV40 large T antigen are responsible for cisplatin resistance through a modulation of DNA repair capacity and/or apoptosis.     

DNA mismatch repair proteins promote apoptosis and suppress tumorigenesis in response to UVB irradiation: an in vivo study

DNA mismatch repair (MMR) proteins are integral to the maintenance of genomic stability and suppression of tumorigenesis due to their role in repair of post-replicative DNA errors. Recent data also support a role for MMR proteins in cellular responses to exogenous DNA damage that does not involve removal of DNA adducts. We have demonstrated previously that both Msh2- and Msh6-null primary mouse embryonic fibroblasts are significantly less sensitive to UVB (ultraviolet B)-induced cytotoxicity and apoptosis than wild-type control cells. In order to ascertain the physiological relevance of the data we have exposed MMR-deficient mice to acute and chronic UVB radiation. We found that MMR-deficiency was associated with reduced levels of apoptosis and increased residual UVB-induced DNA adducts in the epidermis 24-h following acute UVB exposure. Moreover, Msh2-null mice developed UVB-induced skin tumors at a lower level of cumulative UVB exposure and with a greater severity of onset than wild-type mice. The Msh2-null skin tumors did not display microsatellite instability, suggesting that these tumors develop via a different tumorigenic pathway than tumors that develop spontaneously. Therefore, we propose that dysfunctional MMR promotes UVB-induced tumorigenesis through reduced apoptotic elimination of damaged epidermal cells.     

The loss of mdm2 induces p53-mediated apoptosis

The p53 tumor suppressor gene product is negatively regulated by the product of its downstream target, mdm2. The deletion of mdm2 in the mouse results in embryonic lethality at 5.5 days post coitum (d.p.c.) which can be overcome by simultaneous loss of the p53 tumor suppressor, substantiating the importance of the negative regulatory function of MDM2 on p53 function in vivo. Hence, the loss of MDM2 allowed the unregulated p53 protein to continuously exert its growth-suppressing activity, which either led to a complete G1 arrest or induced the p53-dependent apoptotic pathway, resulting in the death of the mdm2-/- embryos. To determine which of these possibilities is occurring, mouse embryo fibroblasts (MEFs) from p53 null and p53/mdm2 double null embryos were transfected with a retroviral vector carrying a temperature-sensitive p53 (tsp53) cDNA. Shifting of single-cell clonal populations to the permissive temperature caused the p53-/-mdm2-/- fibroblasts expressing tsp53 to undergo apoptosis in a dose-dependent manner. This phenotype was not observed in the tsp53 expressing p53-/- clones nor the parental cell lines. Thus, our data indicate that the simple loss of mdm2 can induce the p53-dependent apoptotic pathway in vivo.