Faculty Bibliography

Faithful chromosome segregation is essential for the maintenance of genetic stability during cell division and it is at least partly monitored by the spindle checkpoint, a surveillance mechanism preventing the cell from prematurely entering anaphase. The adenomatous polyposis coli (Apc) gene also plays an important role in regulating genomic stability, as mutations of Apc cause aneuploidy. Here we show that whereas Apc(Min)(/+) mice developed many adenomatous polyps, mostly in the small intestine, by 3 mo of age; BubR1(+/-)Apc(Min)(/+) compound mutant mice developed 10 times more colonic tumors than Apc(Min)(/+) mice. The colonic tumors in BubR1(+/-)Apc(Min)(/+) mice were in higher grades than those observed in Apc(Min)(/+) mice. Consistently, BubR1(+/-)Apc(Min)(/+) murine embryonic fibroblasts (MEFs) contained more beta-catenin and proliferated at a faster rate than WT or BubR1(+/-) MEFs. Moreover, BubR1(+/-)Apc(Min)(/+) MEFs slipped through mitosis in the presence of nocodazole and exhibited a higher rate of genomic instability than that of WT or BubR1(+/-) or Apc(Min)(/+) MEFs, accompanied by premature separation of sister chromatids. Together, our studies suggest that BubR1 and Apc functionally interact in regulating metaphase-anaphase transition, deregulation of which may play a key role in genomic instability and development and progression of colorectal cancer

BACKGROUND: The ATM kinase regulates cell-cycle checkpoints by phosphorylating multiple proteins, including histone H2AX, CHK1, and CHK2 kinases and p53. ATM is activated through auto- or trans- phosphorylation of Ser-1981 in response to DNA damage, particularly induction of DNA double-strand breaks (DSBs). The aim of the present study was to reveal a possible correlation between activation of ATM vis-a-vis H2AX phosphorylation, cell cycle phase, and apoptosis in cells treated with DNA topoisomerase (topo) I (topotecan; Tpt) or topo2 (mitoxantrone; Mtx) inhibitor. MATERIALS AND METHODS: Cultures of HL-60 cells were treated with Tpt or Mtx for various time intervals. ATM or H2AX phosphorylation was detected immunocytochemically, using Ab specific for ATM phosphorylated on Ser-1981 (ATM-S1981(P)) or for H2AX (gammaH2AX) phosphorylated on Ser-139, respectively, concurrent with the analysis of cellular DNA content. Cellular fluorescence was measured by flow cytometry. RESULTS: Untreated cells showed a modest but variable level of labeling with ATM-S1981(P) Ab across the cell cycle, with exception of mitotic cells that were strongly labeled. Exposure of cells to 150 nM Tpt induced ATM phosphorylation concurrent with phosphorylation of H2AX within 10 min; phosphorylation of both proteins was essentially limited to S-phase and was suppressed by caffeine and wortmannin, inhibitors of PI-3-like kinases. Exposure of cells to Mtx also led to ATM and H2AX phosphorylation, which, compared to Tpt, occurred later and was not cell-cycle-phase specific. Apoptosis of HL-60 cells in Tpt or Mtx treated cultures was detected after 2 or 4 h, respectively, and was limited to S-phase cells. CONCLUSIONS: The data are consistent with the role of ATM as a mediator of H2AX phosphorylation in response to DNA damage by topo1 (Tpt) or topo 2 (Mtx) inhibitor. The observed cell-cycle-phase related differences in response to Tpt vs Mtx suggest that while the collisions of DNA replication forks with the 'cleavable complexes' stabilized by topo1 inhibitor are the primary cause of DSBs induced by Tpt, the collisions of RNA polymerase molecules with the complexes stabilized by the topo2 inhibitor play a major role for induction of DSBs by Mtx. The present report is the first that (i) describes cytometric analysis of ATM activation and (ii) demonstrates activation of the enzyme (kinase) and its consequence (substrate phoshorylation), both in relation to cell-cycle phase and onset of apoptosis within the same cells

MEK1, a gene product that regulates cell growth and differentiation, also plays an important role in Golgi breakdown during the cell cycle. We have recently shown that polo-like kinase (Plk3) is Golgi localized and involved in Golgi dynamics during the cell cycle. To study the mode of action of Plk3 in the Golgi fragmentation cascade, we examined functional as well as physical interactions between Plk3 and MEK1/ERKs. In HeLa cells, although a significant amount of Plk3 signals dispersed in a manner similar to those of Golgi during mitosis concentrated Plk3 was detected at spindle poles, which colocalized with phospho-MEKs and phospho-ERKs. Pull-down assays showed that Plk3 physically interacted with MEK1 and ERK2. Nocodazole activated Plk3 and its activation was blocked by MEK-specific inhibitors (PD98059 or U0126). Moreover, transfection of activated MEK1 resulted in an enhanced kinase activity of Plk3; Plk3-induced fragmentation of Golgi stacks was significantly reduced after treatment with MEK inhibitors. Consistently, ectopic expression of activated MEK1, but not kinase-dead MEK1(K97R), stimulated Plk3 to induce Golgi breakdown and the stimulation was not observed in cells expressing Plk3(K52R). Furthermore, PLK3(-/-) murine embryonic fibroblast cells exhibited a significantly less fragmentation of the Golgi complex than that in wild-type cells after exposed to nocodazole. Thus, our studies strongly suggest that Plk3 may be a key protein kinase mediating MEK1 function in the Golgi fragmentation pathway during cell division

A compromised spindle checkpoint is thought to play a key role in genetic instability that predisposes cells to malignant transformation. Loss of function mutations of BubR1, an important component of the spindle checkpoint, have been detected in human cancers. Here we show that BubR1(+/-) mouse embryonic fibroblasts are defective in spindle checkpoint activation, contain a significantly reduced amount of securin and Cdc20, and exhibit a greater level of micronuclei than do wild-type cells. RNA interference-mediated down-regulation of BubR1 also greatly reduced securin level. Moreover, compared with wild-type littermates, BubR1(+/-) mice rapidly develop lung as well as intestinal adenocarcinomas in response to challenge with carcinogen. BubR1 is thus essential for spindle checkpoint activation and tumor suppression

The physiologic function of BUBR1, a key component of the spindle checkpoint, was examined by generating BUBR1-mutant mice. BUBR1(-/-) embryos failed to survive beyond day 8.5 in utero as a result of extensive apoptosis. Whereas BUBR1(+/-) blastocysts grew relatively normally in vitro, BUBR1(-/-) blastocysts exhibited impaired proliferation and atrophied. Adult BUBR1(+/-) mice manifested splenomegaly and abnormal megakaryopoiesis. BUBR1 haploinsufficiency resulted in an increase in the number of splenic megakaryocytes, which was correlated with an increase in megakaryocytic, but a decrease in erythroid, progenitors in bone marrow cells. RNA interference-mediated down-regulation of BUBR1 also caused an increase in polyploidy formation in murine embryonic fibroblast cells and enhanced megakaryopoiesis in bone marrow progenitor cells. However, enhanced megakaryopoiesis in BUBR1(+/-) mice was not correlated with a significant increase in platelets in peripheral blood, which was at least partly due to a defect in the formation of proplatelet-producing megakaryocytes. Together, these results indicate that BUBR1 is essential for early embryonic development and normal hematopoiesis