Endogenous, nontelomeric reverse transcriptase (RT) is encoded by two classes of repeated elements: retrotransposons and endogenous retroviruses. Expression of RT-coding genes is generally repressed in differentiated nonpathological tissues, yet is active in the mammalian germ line, embryonic tissues and tumor cells. Nevirapine is a non-nucleoside RT inhibitor with a well-characterized inhibitory activity on RT enzymes of retroviral origin. Here, we show that nevirapine is also an effective inhibitor of the endogenous RT in murine and human cell lines. In addition, progenitor and transformed cells undergo a significant reduction in the rate of cell growth upon exposure to nevirapine. This is accompanied by the onset of differentiation, as depicted in F9 and C2C7 progenitor cells cultures in which nevirapine triggers the expression of differentiation-specific markers. Consistent with this, an extensive reprogramming of cell cycle gene expression was depicted in nevirapine-treated F9 cultures. Furthermore, nevirapine exposure rescued the differentiation block present in acute myeloid leukemia (AML) cell lines and primary blasts from two AML patients, as indicated by morphological, functional and immunophenotypic assays. The finding that an RT inhibitor can modulate cell proliferation and differentiation suggests that RT may represent a novel target in the development of therapeutical approaches to neoplasia.

Growing evidence indicates a central role for p53 in mediating cell cycle arrest in response to mitotic spindle defects so as to prevent rereplication in cells in which the mitotic division has failed. Here we report that a transient inhibition of spindle assembly induced by nocodazole, a tubulin-depolymerizing drug, triggers a stable activation of p53, which can transduce a cell cycle inhibitory signal even when the spindle-damaging agent is removed and the spindle is allowed to reassemble. Cells transiently exposed to nocodazole continue to express high levels of p53 and p21 in the cell cycle that follows the transient exposure to nocodazole and become arrested in G 1 , regardless of whether they carry a diploid or polyploid genome after mitotic exit. We also show that p53 normally associates with centrosomes in mitotic cells, whereas nocodazole disrupts this association. Together these results suggest that the induction of spindle damage, albeit transient, interferes with the subcellular localization of p53 at specific mitotic locations, which in turn dictates cell cycle arrest in the offspring of such defective mitoses.

Spatial control is a key issue in cell division. The Ran GTPase regulates several fundamental processes for cell life, largely acting through importin molecules. The best understood of these is protein import through the nuclear envelope in interphase, but roles in mitotic spindle assembly are also established. In mammalian cells, in which centrosomes are major spindle organizers, a link is emerging between the Ran network, centrosomes and spindle poles. Here, we show that, after nuclear envelope breakdown, importin β is transported to the spindle poles in mammalian cells. This localization is temporally regulated from prometaphase until anaphase, when importin β dissociates from poles and is recruited back around reforming nuclei. Importin β sediments with mitotic microtubules in vitro and its accumulation at poles requires microtubule integrity and dynamics in vivo. Furthermore, RNA interference-dependent inactivation of TPX2, the major Ran-dependent spindle organizer, abolishes importin β accumulation at poles. Importin β has a functional role in spindle pole organization, because overexpression yields mitotic spindles with abnormal, fragmented poles. Coexpression of TPX2 with importin β mitigates these abnormalities. Together, these results indicate that the balance between importins and spindle regulators of the TPX2 type is crucial for spindle formation. Targeting of TPX2/importin-β complexes to poles is a key aspect in Ran-dependent control of the mitotic apparatus in mammalian cells.

We recently demonstrated that the p53 oncosuppressor associates to centrosomes in mitosis and this association is disrupted by treatments with microtubule-depolymerizing agents. Here, we show that ATM, an upstream activator of p53 after DNA damage, is essential for p53 centrosomal localization and is required for the activation of the postmitotic checkpoint after spindle disruption. In mitosis, p53 failed to associate with centrosomes in two ATM-deficient, ataxiatelangiectasia-derived cell lines. Wild-type ATM gene transfer reestablished the centrosomal localization of p53 in these cells. Furthermore, wild-type p53 protein, but not the p53-S15A mutant, not phosphorylatable by ATM, localized at centrosomes when expressed in p53-null K562 cells. Finally, Ser15 phosphorylation of endogenous p53 was detected at centrosomes upon treatment with phosphatase inhibitors, suggesting that a p53 dephosphorylation step at centrosome contributes to sustain the cell cycle program in cells with normal mitotic spindles. When dissociated from centrosomes by treatments with spindle inhibitors, p53 remained phosphorylated at Ser15. AT cells, which are unable to phosphorylate p53, did not undergo postmitotic proliferation arrest after nocodazole block and release. These data demonstrate that ATM is required for p53 localization at centrosome and support the existence of a surveillance mechanism for inhibiting DNA reduplication downstream of the spindle assembly checkpoint