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Cytokinesis, the final phase of cell division, is necessary to form two distinct daughter cells with correct distribution of genomic and cytoplasmic materials. Its failure provokes genetically unstable states, such as tetraploidization and polyploidization, which can contribute to tumorigenesis. Aurora-B kinase controls multiple cytokinetic events, from chromosome condensation to abscission when the midbody is severed. We have previously shown that HIPK2, a kinase involved in DNA damage response and development, localizes at the midbody and contributes to abscission by phosphorylating extrachromosomal histone H2B at Ser14. Of relevance, HIPK2-defective cells do not phosphorylate H2B and do not successfully complete cytokinesis leading to accumulation of binucleated cells, chromosomal instability, and increased tumorigenicity. However, how HIPK2 and H2B are recruited to the midbody during cytokinesis is still unknown. Here, we show that regardless of their direct (H2B) and indirect (HIPK2) binding of chromosomal DNA, both H2B and HIPK2 localize at the midbody independently of nucleic acids. Instead, by using mitotic kinase-specific inhibitors in a spatio-temporal regulated manner, we found that Aurora-B kinase activity is required to recruit both HIPK2 and H2B to the midbody. Molecular characterization showed that Aurora-B directly binds and phosphorylates H2B at Ser32 while indirectly recruits HIPK2 through the central spindle components MgcRacGAP and PRC1. Thus, among different cytokinetic functions, Aurora-B separately recruits HIPK2 and H2B to the midbody and these activities contribute to faithful cytokinesis.

The p53 oncosuppressor is strictly maintained in an inactive form under normal conditions, while it is post-translationally activated by a variety of stresses, enacting different protective biological functions. Since one critical issue in cancer gene therapy is tumor specificity, we asked whether the tight p53 regulation applies also to exogenously transferred p53. In principle, this type of regulation could allow p53 gene transfer in both normal and tumor cells to produce detrimental effects only in the latter ones. Here, we report that primary bone marrow cells infected with a p53 recombinant retrovirus and transplanted into irradiated mice reconstitute the hematopoietic system, with no detectable alterations in any of its compartments. Furthermore, simultaneous infection of leukemia and bone marrow cells depleted the neoplastic contamination, allowing lifelong, disease-free survival of 65% of the transplanted animals. These results show that exogenous p53 is controlled as tightly as the endogenous one, and opens the way to p53 gene therapy, without requiring tumor targeting.

The E7 oncogene is an essential tool used by papillomaviruses to interfere with the cell cycle and cellular differentiation. We investigated the effects of E7 expression on both cellular functions in skeletal muscle cells, a terminally differentiating system. When expressed in myoblasts, E7 impaired differentiation only partially, but allowed continuation of DNA synthesis during and after differentiation. Surprisingly, E7 expression in terminally differentiated myotubes could not reactivate DNA synthesis even though the oncogene bound the retinoblastoma protein, reduced its levels, and increased E2F transcriptional activity. Despite the high cyclin E protein levels induced by E7, the myotubes remained devoid of cyclin E-associated kinase activity. Enforcement of such activity in the presence of E7 brought myotubes into S phase. These results show that E7, unlike other DNA tumor-virus oncogenes, cannot reactivate the cell cycle in postmitotic myotubes. In contrast, E7 allows significant differentiation to occur in the presence of persisting DNA synthesis. These observations distinguish E7 from other functionally related oncogenes and bear significance for the understanding of the natural life cycle of human papillomaviruses. The fact that E7 alone inhibits the initiation but not the maintenance of the postmitotic state indicates that the mechanisms underlying these two functions are at least partially distinct.

In yeast, MSH2 plays an important role in mismatch repair (MMR) and recombination, whereas the function of the mammalian MSH2 protein in recombinational repair is not completely established. We examined the cellular responses of MSH2-deficient mouse cells to X-rays to clarify the role of MSH2 in recombinational repair. Cell survival, checkpoint functions and relocalization of the recombination-related proteins MRE11 and RAD51 were analysed in embryonic fibroblasts derived from MSH2(+/+) and MSH2(-/-) mice, and in MSH2-proficient and deficient mouse colorectal carcinoma cells. Loss of MSH2 function was found to be associated with reduction in cell survival following radiation, absence of either MRE11 or RAD51 relocalization and a higher level of X-ray-induced chromosomal damage specifically in G2-phase cells. Finally, MSH2(-/-) cells showed an inefficient early G2/M checkpoint, being arrested only transiently after irradiation before progressing into mitosis. Consistent with the premature release from the G2-phase arrest, activation of CHK1 was transient and CHK2 was not phosphorylated in synchronized MSH2-null cells. Our data suggest that an active MSH2 is required for a correct response to ionizing radiation-induced DNA damage in the G2 phase of the cell cycle, possibly connecting DSB repair to checkpoint signalling.

We have previously shown that the adenovirus E1A oncogene can reactivate the cell cycle in terminally differentiated cells. Current models imply that much or all of this E1A activity is mediated by the release of the E2F transcription factors from pocket-protein control. In contrast, we show here that overexpression of E2F-1, E2F-2 and E2F-4, or a chimeric E2F-4 tethered to a nuclear localization signal cannot reactivate postmitotic skeletal muscle cells (myotubes). This is not due to lack of transcriptional activity, as demonstrated on both a reporter construct and a number of endogenous target genes. Although cyclin E was strongly overexpressed in E2F-transduced myotubes, it lacked associated kinase activity, possibly explaining the inability of the myotubes to enter S phase and accumulate cyclin A. Although E2F is not sufficient to trigger DNA synthesis in myotubes, its activity is necessary even in the presence of E1A, as dominant-negative DP-1 mutants inhibit E1A-mediated cell cycle reentry. Our data show that, to reactivate myotubes, E1A must exert other functions, in addition to releasing E2F. They also establish mouse myotubes as an experimental system uniquely suited to study the most direct E2F functions in the absence of downstream cell cycle effects.

Expression of exogenous wt-p53 in different tumor cell lines can induce growth arrest, apoptosis, or differentiation. Several experimental works have highlighted the relevance of cellular context in the determination of p53-mediated final outcomes. We recently observed that these diverse wt-p53 effects can also be induced by overexpressing wt-p53 in a single cell type-the 32D myeloid progenitors-transformed with different activated oncogenes. Here we show that 32D cells transformed with two different oncogenes, v-src or c-fms [S301,F969], both belonging to the CSF-1 transduction pathway, respond to exogenous wt-p53 expression with the same final outcome-monocytic differentiation. This result is particularly significant since 32D cells do not spontaneously express the CSF-1 receptor, whereas they undergo granulocytic differentiation upon G-CSF stimulation. These data strongly support the idea that wt-p53 suppressing effects result from interactions between p53 activity and the signaling pathways activated in different transformed cells.

Alterations of the tumor suppressor gene p53 are uncommon in differentiated thyroid neoplasia but are detected at high frequency in anaplastic thyroid carcinoma suggesting that impaired p53 function may contribute to the undifferentiated and highly aggressive phenotype of these tumors. Effects of wild type p53 (wt-p53) re-expression were investigated in a human anaplastic thyroid carcinoma cell line (ARO) expressing a mutated p53. ARO cells were stably transfected with the temperature-sensitive p53 Val135 gene (ts-p53) which exhibits wild type-like activity at 32 degrees C. Exogenous wt-p53 function in ARO-tsp53 clones was assessed by evaluating its transcriptional activity on a CAT reporter vector containing p53 binding sites. At 32 degrees C, a significant reduction in the proliferation rate (approximately or equal to 50%) was observed, with accumulation of cells in the G0/G1 phase of the cell cycle. This effect was accompanied by induction of the expression of the growth inhibitor p21/Waf1 gene. At 32 degrees C, ARO-tsp53 clones also showed a marked impairment of their tumorigenic potential. Furthermore, transfected clones re-acquired the ability to respond to thyrotropin (TSH) stimulation showing an increased expression of thyroid-specific genes (thyroglobulin, thyroperoxidase and TSH receptor). In conclusion, re-expression of wt-p53 activity in ARO cells, inhibits cell proliferation and restores responsiveness to physiological stimuli.

Cyclin Bl plays an important role in cell proliferation. Its expression is tightly regulated at the mRNA and protein levels during the cell cycle and is found to be deregulated in various malignancies. To enlighten the signalling pathways which lead to the cell cycle dependent expression of the cyclin B1 gene, we studied its transcriptional regulation in quiescent and proliferating NIH3T3 cells. We previously showed that the transcriptional activity of the cyclin B1 promoter decreases in quiescent cells. Here, we map a quiescence-responsive element of the human cyclin B1 promoter to an E-box sequence, CACGTG, which spans positions -124/-119. Nuclear proteins protect this sequence in a DNase I digestion assay and bind, in electromobility shift assays, an oligonucleotide spanning positions -133/-110. Max-specific antibodies block the DNA-binding activity of protein complexes to this probe. A mutation in the E-box core sequence abolishes the decrease in transcription that occurs during quiescence. Finally, we find that over-expression of Max protein in proliferating cells specifically inhibits cyclin B1 promoter activity through this E-box. Moreover, Max over-expression in proliferating NIH3T3 cells leads to down-regulation of the endogenous cyclin B1 protein. In conclusion, these data support a model whereby E-box-binding proteins mediate the decrease in the transcriptional activity of the cyclin B1 promoter observed in quiescent cells and suggest that Max contributes to this response.

Apoptotic cell death is an active process which regulates the maintenance of the hematopoietic homeostasis. It has been reported that wild-type p53 (wt-p53) protein induces apoptosis in leukemia cells. To assess whether p53 is involved in the apoptotic process of normal hematopoietic cells, we introduced the temperature-sensitive p53Val135 mutant into the murine myeloid precursor cell line 32Dcl3. These are diploid, non-tumorigenic cells whose survival and proliferation are dependent upon growth factor supply (IL-3 and serum). Overexpression of wt-p53 protein does not affect morphology and proliferation of 32D cells as long as they are maintained in the presence of IL-3. However, after IL-3 withdrawal, wt-p53 overexpression significantly accelerates apoptosis. This phenomenon is IL-3 specific since no differences in death rates induced by serum starvation are found between parental cells and p53-transfectants. When the latter experiments are carried out at 37 degrees C with p53 protein in mutant conformation, an extended survival of 32D cells is observed after IL-3 deprivation, but not after serum withdrawal. Taken together, these results show that wt-p53 actively mediates the apoptosis due to the absence of specific growth factors, such as IL-3, suggesting that p53 might be involved in the response of myeloid precursors to environmental cytokines for the maintenance of the hematopoietic homeostasis.