_:b6279403 . _:b416968224 . _:b416968246 "2"^^ . _:b6279384 "We also quantified unspliced, intron-containing Lats2 precursor mRNA, expected to more reliably capture differences in transcription rates (Phelps et al. >>2006<<; Ponzio et al. 2007)." . _:b416968111 "9"^^ . _:b416968241 "2"^^ . . _:b416968240 "2"^^ . _:b416968243 "2"^^ . _:b6279390 . . . _:b416968242 "2"^^ . _:b416968253 "2"^^ . _:b416968194 . _:b416968252 "2"^^ . . _:b6279375 "Using a system of V-Ras-transformed NIH3T3, Li et al (>>2003<<) found that overexpression of Lats2 could suppress tumorigenesis in nude mice." . _:b416968117 "5"^^ . _:b416968254 "2"^^ . _:b6279414 "5D and the fact that the WI-38/hTERTfast cells underwent extensive passaging in vitro (Milyavsky et al. >>2003<<) prior to H-RasV12 infection, it appears more likely that the stress response elicited through activation of the ATR-Chk1-Lats2-p53 pathway selects for cells with preexisting Lats2 promoter hypermethylation, rather than inducing multiple" . _:b416968116 "5"^^ . _:b416968249 "2"^^ . . _:b416968119 "4"^^ . _:b416968248 "2"^^ . _:b416968118 "4"^^ . _:b6279413 "A number of studies suggest that oncogenic Ras can induce the expression of several DNA methyltransferases (Pruitt et al. 2005; Chang et al. >>2006<<), hence, it is possible that the silencing of Lats2 occurs only after H-RasV12 introduction." . _:b416968251 "2"^^ . _:b416968113 "5"^^ . _:b416968250 "2"^^ . _:b416968112 "5"^^ . _:b416968218 . _:b6279397 "Furthermore, as previously observed (Denko et al. >>1995<<; Abulaiti et al. 2006), cells expressing oncogenic H-RasV12 tended to form micronuclei; these micronuclei contained Lats2 (arrowhead)." . _:b416968115 "5"^^ . _:b416968114 "5"^^ . _:b416968220 . _:b416968125 "3"^^ . _:b416968124 "4"^^ . _:b416968127 "3"^^ . . _:b416968126 "3"^^ . _:b416968157 . _:b416968123 . _:b416968121 "4"^^ . . _:b416968144 . . _:b416968145 . _:b416968213 . _:b416968146 . _:b416968120 "4"^^ . _:b416968147 . _:b416968148 . _:b416968149 . _:b416968150 . . _:b416968123 "4"^^ . _:b416968151 . _:b416968152 . _:b416968153 . _:b416968154 . _:b416968122 "4"^^ . _:b416968125 . _:b416968155 . _:b416968156 . _:b416968157 . _:b416968138 . _:b416968133 "3"^^ . _:b416968158 . _:b416968155 . _:b416968159 . _:b6279422 "We propose that generation of mitotic stress, either by treatment with nocodazole (Aylon et al. >>2006<<) or oncogene activation (this work) is necessary for Lats2 nuclear import." . _:b416968128 . _:b416968129 . _:b416968132 "3"^^ . _:b416968130 . _:b416968200 . _:b416968131 . _:b416968132 . _:b416968133 . . _:b416968135 "3"^^ . _:b416968183 . _:b416968134 . _:b416968135 . _:b416968136 . _:b416968137 . _:b416968205 . _:b416968134 "3"^^ . _:b416968138 . _:b416968139 . _:b416968140 . _:b416968141 . _:b416968129 "3"^^ . _:b416968142 . _:b416968143 . _:b416968176 . _:b6279425 "treatment of astrocytoma cell lines with a DNA methyltransferase inhibitor restored Lats2 levels and caused apoptosis, indicating that silencing of Lats2 is an important step in tumor cell resistance to apoptosis (Jiang et al. >>2006<<). Downregulation of Lats2 via promoter hypermethylation is also associated with poor prognosis in acute lymphoblastic leukemia (Jimenez-Velasco et al. 2005)." . . . . _:b416968128 "3"^^ . _:b416968177 . _:b416968178 . _:b416968179 . _:b416968180 . _:b416968181 . . _:b416968131 "3"^^ . _:b416968182 . _:b416968183 . _:b416968184 . . _:b416968185 . _:b416968130 "3"^^ . _:b416968177 . _:b416968186 . _:b416968187 . _:b416968188 . _:b416968188 . _:b416968189 . _:b416968141 "3"^^ . _:b416968190 . . _:b416968191 . _:b6279392 "ATR-mediated phosphorylation of the Chk1 kinase on serine 345 is key to the propagation of replication stress signals (Jiang et al. >>2003<<). Indeed, oncogenic H-Ras led to a substantial induction of Chk1 Ser345 phosphorylation, suggested by the appearance of a slower migrating form of Chk1 and formally proven with phosphospecific antibodies (Fig 2A, compare lanes 1 and 5)." . _:b416968160 . _:b416968161 . _:b416968140 "3"^^ . _:b416968162 . _:b416968163 . _:b416968164 . _:b416968165 . _:b416968143 "3"^^ . _:b416968166 . _:b416968167 . _:b416968168 . _:b416968169 . _:b416968142 "3"^^ . _:b416968170 . _:b416968171 . _:b416968172 . _:b416968173 . . _:b416968137 "3"^^ . _:b416968111 . _:b416968174 . _:b416968175 . _:b416968243 . _:b416968136 "3"^^ . _:b416968139 "3"^^ . . _:b416968138 "3"^^ . _:b6279373 "Even in those cancers that retain wild-type p53, its activity is likely to be compromised by genetic or epigenetic deficiencies in upstream pathways (Vazquez et al. 2008; Wang and El-Deiry >>2008<<)." . _:b416968149 "3"^^ . _:b416968148 "3"^^ . _:b416968208 . _:b416968142 . _:b416968120 . _:b416968151 "2"^^ . _:b416968117 . . . _:b416968116 . _:b416968235 . _:b416968150 "3"^^ . _:b416968186 . _:b6279404 . _:b416968229 . _:b416968119 . _:b416968145 "3"^^ . _:b416968112 . _:b416968143 . _:b416968113 . . _:b416968144 "3"^^ . _:b416968118 . _:b416968114 . _:b416968115 . _:b416968116 . . _:b416968117 . _:b416968147 "3"^^ . _:b416968113 . _:b416968118 . _:b416968119 . _:b416968120 . _:b416968121 . . _:b416968146 "3"^^ . _:b416968146 . _:b416968112 . _:b416968122 . _:b416968123 . _:b416968124 . _:b416968157 "2"^^ . _:b416968125 . _:b416968115 . _:b416968126 . _:b6279385 . _:b6279371 "More recent evidence implicates Ras-induced p53-dependent senescence as a pivotal cancer-inhibitory mechanism also in animal models and in humans (Bartek et al. 2007; Ventura et al. 2007; Xue et al. 2007; Halazonetis et al. >>2008<<). Consistent with this view, the tumor suppressor p53 is mutated at a high frequency in many tumor types. Even in those cancers that retain wild-type p53, its activity is likely to be compromised by genetic or epigenetic deficiencies in" . . . _:b416968127 . _:b416968137 . _:b416968156 "2"^^ . _:b416968114 . . _:b416968159 "2"^^ . _:b416968117 . _:b416968125 . _:b416968129 . _:b6279383 . _:b416968187 . _:b6279391 "On the other hand the ATM-related kinase, ATR, known to play an important role in the response to DNA replication stress, has also been implicated in H-Ras signaling (Abulaiti et al. 2006; Fikaris et al. 2006; Mallette et al. >>2007<<). We therefore investigated whether ATR might be involved in relaying the H-Ras-driven signal for Lats2 induction. Indeed, expression of H-RasV12 caused a marked increase of phosphorylated ATR (Fig 2A, lanes 1,5), confirming that" . _:b416968158 "2"^^ . _:b416968124 . _:b416968190 . _:b6279375 . _:b416968161 . _:b416968153 "2"^^ . _:b416968127 . _:b416968111 . _:b6279409 "Moreover, Lats2 promoter hypermethylation in tumors has been documented (Jiang et al. >>2006<<; Shema et al. 2008)." . _:b416968152 "2"^^ . _:b416968126 . . _:b416968155 "2"^^ . _:b416968121 . _:b416968154 "2"^^ . _:b416968120 . _:b416968225 . _:b6279365 . _:b6279393 . _:b6279376 . _:b416968165 "2"^^ . _:b6279395 "Lats2 is predominantly a centrosomal protein (Toji et al. >>2004<<). Upon damage to the mitotic apparatus Lats2 departs from the centrosome and accumulates in the nucleus, where it binds Mdm2 and stabilizes p53 (Aylon et al. 2006)." . _:b416968123 . _:b416968241 . _:b416968164 "2"^^ . _:b416968122 . _:b6279405 "5A, fourth row), unlike the typical response (Aylon et al. >>2006<<; see also Fig. 5A, second row)." . _:b416968168 . _:b416968167 "2"^^ . _:b416968133 . . _:b6279377 . _:b416968192 . _:b416968166 "2"^^ . . . _:b416968132 . . _:b6279385 "We also quantified unspliced, intron-containing Lats2 precursor mRNA, expected to more reliably capture differences in transcription rates (Phelps et al. 2006; Ponzio et al. >>2007<<). This analysis revealed a nearly 4.5 fold increase in Lats2 precursor mRNA in response to H-RasV12 expression (Fig 1B, gray bars). A similar trend was observed also in non-immortalized WI-38 cells (Supplementary Fig S1). Hence, oncogenic" . _:b416968161 "2"^^ . _:b6279382 . _:b416968135 . _:b416968166 . _:b416968160 "2"^^ . _:b416968134 . _:b6279386 . _:b416968163 "2"^^ . _:b416968129 . _:b416968126 . _:b416968116 . _:b416968162 "2"^^ . _:b416968128 . _:b416968173 "2"^^ . _:b416968131 . _:b416968172 "2"^^ . _:b416968130 . . _:b416968175 "2"^^ . _:b416968141 . . . _:b416968174 "2"^^ . _:b416968147 . _:b6279411 . _:b416968140 . _:b416968169 "2"^^ . _:b416968143 . _:b416968226 . . _:b416968168 "2"^^ . . _:b416968142 . _:b416968171 "2"^^ . _:b416968137 . _:b416968170 "2"^^ . _:b416968136 . . _:b416968113 . . _:b416968181 "2"^^ . _:b416968139 . _:b416968191 . _:b416968135 . _:b416968180 "2"^^ . _:b416968138 . _:b416968183 "2"^^ . _:b416968149 . . . _:b416968182 "2"^^ . _:b416968148 . _:b416968177 "2"^^ . _:b416968151 . _:b416968165 . _:b6279368 "More recent evidence implicates Ras-induced p53-dependent senescence as a pivotal cancer-inhibitory mechanism also in animal models and in humans (Bartek et al. >>2007<<; Ventura et al. 2007; Xue et al. 2007; Halazonetis et al. 2008)." . _:b416968176 "2"^^ . _:b416968150 . _:b6279393 "It is also noteworthy that both these cell types lack functional p14ARF tumor suppressor protein (Burri et al. >>2001<<; Milyavsky et al. 2003), indicating that, at least in WI-38 and HCT116 cells, ARF is dispensable for the induction of Lats2 by oncogenic Ras." . _:b416968179 "2"^^ . _:b416968145 . . _:b416968201 . _:b416968178 "2"^^ . _:b416968144 . _:b416968189 "2"^^ . . _:b416968147 . _:b416968188 "2"^^ . . _:b416968146 . _:b416968191 "2"^^ . _:b6279415 . _:b6279410 "Moreover, Lats2 promoter hypermethylation in tumors has been documented (Jiang et al. 2006; Shema et al. >>2008<<). We therefore asked whether elevated Lats2 promoter hypermethylation might underlie also the downregulation of Lats2 transcription in this in vitro \u201Ctumor progression\u201D model. To this end, we performed methylation-specific qPCR analysis" . _:b416968157 . . _:b416968190 "2"^^ . _:b416968202 . _:b416968245 . _:b416968210 . _:b416968156 . _:b6279414 . _:b6279407 "Remarkably, once cells had switched off the Lats2 axis, even reintroduction of extra H-RasV12 failed to upregulate Lats2 again, despite a clear increase in H-Ras protein (Fig 5D, compare lane 4 and 6). Likewise, Abulaiti et al. (>>2006<<) reported that chronic overexpression of Ras in cultured rat thyroid cells selects for the emergence of survivors that have switched off the apoptotic response to excess Ras; such cells were shown to display compromised p53 activity and to" . _:b416968185 "2"^^ . _:b416968159 . _:b6279401 "Like apoptosis, senescence serves as a barrier to prevent oncogene-driven cellular transformation (Lowe >>1999<<; Bartkova et al. 2006; Kim et al. 2007)." . . . . _:b416968184 "2"^^ . _:b416968158 . _:b416968187 "2"^^ . _:b416968153 . _:b416968186 "2"^^ . _:b416968152 . _:b416968155 . _:b6279366 . _:b6279386 "The microRNA miR-373 can target directly Lats2 mRNA (Voorhoeve et al. >>2006<<). Indeed, H-RasV12 overexpression downregulated miR-373 levels (Supplementary Fig S2), suggesting that repression of miR-373 expression by activated H-Ras also might, in principle, promote Lats2 induction." . . . _:b416968154 . _:b416968176 . _:b416968165 . . _:b416968164 . . _:b416968167 . . _:b416968166 . _:b416968161 . _:b416968160 . _:b416968215 . _:b416968163 . . _:b416968162 . _:b416968130 . . _:b6279416 . . _:b416968173 . _:b416968172 . _:b416968134 . _:b416968175 . . _:b416968174 . . _:b6279381 "Lats2 was detected with anti-KPM/Lats2 (Santa Cruz) or LA-2 (Yabuta et al. >>2007<<). Protein signals were quantified using ImageJ software and normalized to the corresponding GAPDH signals." . _:b6279366 "Activating mutations in the Ras pathway occur in approximately 30% of all human cancers (Ding et al. >>2008<<). However, mutation of Ras alone is insufficient to induce tumor development. To a large measure, this is due to the fact that aberrant activation of oncogenic Ras triggers a series of protective cellular responses aimed at curtailing its" . _:b416968169 . _:b6279367 . _:b416968169 . . _:b6279392 . . . _:b416968168 . _:b6279394 . _:b416968236 . . _:b416968171 . _:b416968240 . _:b416968170 . _:b416968181 . . . _:b6279365 . _:b416968180 . _:b416968207 . _:b416968133 . _:b6279401 . _:b416968212 . _:b6279366 . _:b416968183 . . _:b6279367 . _:b416968182 . _:b416968239 . _:b416968177 . _:b416968244 . _:b416968176 . . . . _:b416968179 . . _:b6279398 "Furthermore, as previously observed (Denko et al. 1995; Abulaiti et al. >>2006<<), cells expressing oncogenic H-RasV12 tended to form micronuclei; these micronuclei contained Lats2 (arrowhead)." . _:b416968178 . _:b416968174 . _:b416968231 . _:b6279372 . _:b416968189 . _:b6279373 . _:b416968188 . _:b6279380 "Immunofluorescence was performed as described (Aylon et al. >>2006<<). Lats2 was detected with anti-KPM/Lats2 (Santa Cruz) or LA-2 (Yabuta et al. 2007)." . _:b6279374 . _:b6279408 . _:b416968191 . _:b416968152 . _:b6279375 . _:b416968190 . . _:b6279368 . _:b416968185 . _:b416968254 . _:b416968227 . _:b6279369 . _:b416968184 . _:b6279370 . . _:b416968187 . "PMC0" . _:b6279371 . _:b416968186 . . _:b6279380 . _:b6279381 . _:b416968170 . _:b6279382 . . _:b416968154 . _:b6279383 . _:b416968111 . _:b6279376 . _:b6279377 . . . _:b6279421 . _:b6279378 . _:b6279379 . . _:b6279388 . _:b6279389 . _:b416968228 . . . _:b6279390 . _:b6279409 . _:b416968122 . _:b416968208 . _:b6279391 . _:b416968209 . _:b416968210 . . _:b416968211 . _:b416968212 . _:b6279384 . _:b416968223 . _:b416968213 . _:b416968132 . _:b416968214 . _:b416968215 . . _:b6279385 . _:b416968216 . _:b416968217 . _:b416968218 . _:b416968219 . _:b6279399 . _:b416968189 . _:b6279386 . _:b416968220 . _:b416968221 . _:b416968222 . _:b416968223 . _:b6279396 . _:b416968193 . _:b6279387 . _:b416968192 . _:b416968193 . _:b416968194 . _:b416968222 . _:b416968195 . . _:b6279396 . _:b6279395 . _:b416968196 . _:b416968197 . _:b416968198 . _:b416968199 . _:b416968200 . _:b6279397 . _:b416968233 . . _:b416968201 . _:b416968202 . _:b416968203 . . _:b6279398 . _:b416968204 . . _:b416968205 . _:b416968206 . _:b416968207 . _:b416968240 . _:b6279399 . _:b416968241 . . _:b416968242 . _:b416968243 . _:b416968244 . _:b6279392 . _:b416968245 . . _:b416968246 . _:b416968247 . _:b416968248 . _:b6279393 . _:b416968249 . . _:b6279426 "Downregulation of Lats2 via promoter hypermethylation is also associated with poor prognosis in acute lymphoblastic leukemia (Jimenez-Velasco et al. >>2005<<). Furthermore, 50% of non-small cell lung carcinoma tumors exhibit decreased Lats2 expression (Strazisar et al. 2008). Interestingly, diminished Lats2 levels are highly correlated with lower expression of the p53 target gene Mdm2" . _:b416968250 . _:b416968251 . _:b416968252 . _:b6279394 . _:b416968253 . _:b416968254 . _:b416968224 . _:b6279395 . _:b416968225 . _:b416968226 . _:b416968227 . _:b6279405 . _:b416968228 . _:b6279404 . . _:b416968229 . _:b416968230 . _:b416968231 . _:b416968232 . _:b6279405 . _:b416968233 . _:b416968234 . _:b416968235 . _:b416968236 . _:b6279406 . _:b416968237 . _:b416968238 . _:b416968239 . _:b6279400 . _:b6279407 . _:b6279400 . _:b6279401 . _:b6279402 . _:b416968248 . _:b6279403 . _:b6279411 _:b6279424 . _:b6279412 . _:b6279411 _:b6279425 . _:b6279411 _:b6279426 . _:b6279413 . _:b6279371 . . _:b6279388 "However, Lats2 is not activated in response to overt DNA damage (Aylon et al. >>2006<<). On the other hand the ATM-related kinase, ATR, known to play an important role in the response to DNA replication stress, has also been implicated in H-Ras signaling (Abulaiti et al. 2006; Fikaris et al. 2006; Mallette et al. 2007). We" . _:b6279414 . _:b6279415 . _:b416968150 . _:b6279408 . _:b416968179 . _:b6279422 . . _:b6279409 . . _:b6279410 . _:b416968185 . . . _:b6279420 . _:b416968124 . . . _:b6279411 . . _:b6279420 . . . _:b6279421 . _:b6279419 . . _:b6279422 . _:b416968114 . _:b6279423 . . _:b6279402 . _:b6279416 . _:b6279418 "Furthermore, by creating centrosome dysfunction (McDermott et al. >>2006<<), prior loss of p16 might facilitate Lats2 activation through chronic mitotic apparatus stress (Aylon et al. 2006)." . _:b6279417 . . _:b6279370 . _:b6279418 . _:b416968238 . . _:b6279419 . _:b6279396 "Upon damage to the mitotic apparatus Lats2 departs from the centrosome and accumulates in the nucleus, where it binds Mdm2 and stabilizes p53 (Aylon et al. >>2006<<). Remarkably, nuclear accumulation of Lats2, which occurs only rarely and sporadically in unperturbed WI-38/hTERTfast cells (Fig 2B, vector/mock), was dramatically elicited by transient overexpression of H-RasV12 (Fig 2B, H-RasV12/mock)." . . . _:b416968211 . . . _:b6279369 . _:b6279372 "Even in those cancers that retain wild-type p53, its activity is likely to be compromised by genetic or epigenetic deficiencies in upstream pathways (Vazquez et al. >>2008<<; Wang and El-Deiry 2008)." . . _:b416968209 . _:b416968182 . . _:b6279399 "In response to mitotic apparatus damage, Lats2 stabilizes p53 by alleviating Mdm2-dependent p53 ubiquitylation and degradation (Aylon et al. >>2006<<). Likewise, oncogenic activation also caused Lats2-dependent p53 accumulation. Whereas knockdown of Lats2 did not affect basal p53 levels (Fig." . _:b416968214 . _:b416968164 . _:b6279374 . . . . _:b416968128 . _:b6279377 "materials and methods" . _:b6279373 . _:b416968232 . _:b6279408 "Promoter hypermethylation plays a significant role in gene inactivation in various human cancers (reviewed in Esteller >>2008<<). Moreover, Lats2 promoter hypermethylation in tumors has been documented (Jiang et al. 2006; Shema et al. 2008)." . _:b416968203 . _:b416968230 . _:b6279397 . _:b6279413 . _:b416968156 . . _:b416968131 . _:b416968145 . . _:b6279417 "Notably, loss of p16(INK4A) expression, which took place in the course of WI-38/hTERTfast cell propagation in culture (Milyavsky et al. 2003), may have accelerated DNA methylation (Reynolds et al. >>2006<<), including that of the Lats2 promoter, thus generating a larger pool of cells to select from in response to H-RasV12-induced stress." . _:b6279380 . _:b6279383 "oncogenic H-Ras activation and Lats2, we made use of WI-38 human lung embryonic fibroblasts that had been immortalized with telomerase and subsequently subjected to long term passaging in culture (WI-38/hTERTfast; Milyavsky et al. >>2003<<). Four days after infection of these cells with a recombinant retrovirus encoding constitutively activated mutant H-RasV12, a marked increase in endogenous Lats2 protein was evident (Fig 1A)." . . _:b6279378 . . _:b416968160 . _:b6279376 "Subsequently, Voorhoeve et al (>>2006<<) reported that downregulation of Lats2 via overexpression of miR-372/3 could bypass H-Ras-induced senescence in primary human fibroblasts." . . _:b416968149 . . _:b6279388 . _:b6279406 "As previously reported (Abulaiti et al. >>2006<<), long-term H-RasV12 expressors displayed an elevated incidence of micronuclei (Fig 5A, DAPI), which became even more pronounced following nocodazole treatment." . _:b416968217 . _:b6279417 . . . _:b416968115 . . _:b6279410 . _:b416968180 . _:b6279411 "discussion" . _:b416968171 . . _:b416968197 . _:b6279365 "introduction" . _:b416968151 . _:b416968221 . _:b416968196 . . _:b416968112 . _:b416968199 . . _:b416968184 . . . _:b416968198 . . _:b416968193 . _:b416968192 . . . _:b416968195 . _:b416968194 . _:b416968205 . . _:b416968247 . _:b416968204 . _:b416968153 . _:b416968195 . _:b416968207 . . _:b416968206 . . . _:b416968201 . _:b6279370 "More recent evidence implicates Ras-induced p53-dependent senescence as a pivotal cancer-inhibitory mechanism also in animal models and in humans (Bartek et al. 2007; Ventura et al. 2007; Xue et al. >>2007<<; Halazonetis et al. 2008)." . _:b416968200 . _:b6279389 "On the other hand the ATM-related kinase, ATR, known to play an important role in the response to DNA replication stress, has also been implicated in H-Ras signaling (Abulaiti et al. >>2006<<; Fikaris et al. 2006; Mallette et al. 2007)." . _:b6279415 "silencing of the INK4A/ARF tumor suppressor locus by promoter hypermethylation in seemingly normal tissue might provide the setting for the emergence of tumor initiating cells upon subsequent oncogene activation (Bean et al. >>2007<<). Thus, it is tempting to speculate that, likewise, cells with silenced Lats2 may also be present sporadically in normal tissue, offering ready soil for oncogene-driven transformation and tumor development." . . . _:b416968203 . _:b416968127 . _:b416968202 . _:b6279365 _:b6279376 . _:b416968199 . _:b6279423 "Thus, in breast cancers, decreased Lats2 expression is linked to biologically aggressive phenotypes, such as large tumor size, high lymph node metastasis and estrogen- and progresterone-receptor negativity (Takahashi et al. >>2005<<). Similarly, in astrocytoma, hypermethylation of the Lats2 promoter was detected in over 70% of the cases and associated with higher tumor grade and increased overall malignancy (Jiang et al. 2006). Moreover, treatment of astrocytoma cell" . _:b416968213 . _:b416968212 . . _:b6279391 . _:b416968250 . . _:b6279400 "Interestingly, similar to what has been observed in cells under mitotic apparatus stress (Aylon et al. >>2006<<), induction of Lats2 mRNA by oncogenic H-Ras was entirely dependent on p53 (Fig 3D)." . _:b6279424 . _:b416968215 . . _:b6279425 . _:b416968214 . _:b416968209 . . _:b416968234 . _:b6279412 . _:b6279365 _:b6279366 . . _:b6279365 _:b6279367 . _:b416968208 . . _:b6279365 _:b6279368 . _:b6279365 _:b6279369 . _:b6279365 _:b6279370 . _:b416968141 . _:b416968119 . _:b6279365 _:b6279371 . _:b416968211 . _:b6279365 _:b6279372 . _:b416968139 . _:b6279365 _:b6279373 . _:b6279365 _:b6279374 . _:b6279365 _:b6279375 . _:b416968210 . _:b416968221 . _:b416968220 . _:b416968223 . _:b6279382 _:b6279408 . _:b416968246 . . _:b6279382 _:b6279409 . _:b6279382 _:b6279410 . _:b416968222 . _:b6279420 "3D); this is reminiscent of the response to mitotic apparatus stress, where p53 is also required for induction of Lats2 gene expression (Aylon et al. >>2006<<). On the other hand, the Ras-ATR-Chk1 axis alters the stability and subcellular localization of the Lats2 protein. Activated H-Ras triggers the departure of Lats2 from centrosomes and its subsequent translocation into the nucleus; whereas" . _:b6279382 _:b6279404 . . _:b6279382 _:b6279405 . _:b6279382 _:b6279406 . _:b6279382 _:b6279407 . _:b416968217 . _:b6279382 _:b6279400 . _:b6279382 _:b6279401 . . _:b6279382 _:b6279402 . _:b416968242 . _:b6279382 _:b6279403 . _:b416968216 . _:b416968140 . . _:b6279382 _:b6279396 . _:b6279382 _:b6279397 . _:b6279382 _:b6279398 . _:b6279382 _:b6279399 . _:b6279390 "On the other hand the ATM-related kinase, ATR, known to play an important role in the response to DNA replication stress, has also been implicated in H-Ras signaling (Abulaiti et al. 2006; Fikaris et al. >>2006<<; Mallette et al. 2007)." . _:b416968219 . . _:b6279382 _:b6279392 . _:b6279382 _:b6279393 . _:b416968216 . _:b416968181 . _:b6279382 _:b6279394 . _:b6279382 _:b6279395 . _:b6279379 "Western blot analysis was performed using standard procedures, employing the following antibodies: Lats2 (LA-2; Yabuta et al. >>2007<<), GAPDH (Chemicon), H-Ras (sc-520, Santa Cruz); p53 (mix of DO1+PAb1801) and p21 (sc-397, Santa Cruz)." . _:b416968218 . _:b6279382 _:b6279388 . _:b6279382 _:b6279389 . . _:b6279382 _:b6279390 . _:b6279382 _:b6279391 . _:b416968229 . _:b6279382 _:b6279384 . _:b6279382 _:b6279385 . "10.1038%2Fonc.2009.270" . _:b6279382 _:b6279386 . _:b6279382 _:b6279387 . _:b416968228 . . _:b6279382 _:b6279383 . _:b416968231 . _:b416968163 . _:b6279368 . _:b6279389 . _:b416968230 . _:b6279377 _:b6279378 . _:b6279377 _:b6279379 . _:b416968225 . _:b6279377 _:b6279380 . _:b6279377 _:b6279381 . _:b416968224 . _:b416968227 . . _:b6279369 "More recent evidence implicates Ras-induced p53-dependent senescence as a pivotal cancer-inhibitory mechanism also in animal models and in humans (Bartek et al. 2007; Ventura et al. >>2007<<; Xue et al. 2007; Halazonetis et al. 2008)." . _:b416968226 . _:b416968175 . _:b416968237 . . _:b416968236 . _:b6279421 "Perhaps relevant to this notion is the observation by Toji et al. (>>2004<<) that preventing the phosphorylation of Lats2 by Aurora A kinase releases Lats2 from the centrosome, but is not sufficient for nuclear accumulation." . _:b6279403 "Like apoptosis, senescence serves as a barrier to prevent oncogene-driven cellular transformation (Lowe 1999; Bartkova et al. 2006; Kim et al. >>2007<<). Previously, Lats2 was found as a target of miR-372/3 in a screen for bypassing H-RasV12-induced, p53-dependent senescence in primary BJ cells (Voorhoeve et al. 2006), suggesting that Lats2 may mediate the H-RasV12 signal that triggers" . _:b6279398 . _:b416968239 . . . _:b416968238 . . . _:b416968233 . _:b416968197 "2"^^ . _:b6279378 "qRT-PCR was performed as described (Aylon et al. >>2006<<). For small RNAs (5S and miR-373), 2.5 \u03BCg total RNA was converted to cDNA using specific primers for reverse transcription (Ambion) and MMLV reverse transcriptase." . . . _:b416968232 . _:b416968196 "2"^^ . _:b416968235 . _:b416968199 "2"^^ . . _:b416968234 . _:b416968198 "2"^^ . _:b416968245 . _:b416968193 "2"^^ . _:b6279387 "recently have been proposed to mediate cellular senescence in response to DNA damage occurring downstream to aberrant oncogene activation, thereby setting a barrier against oncogene-driven transformation (reviewed in Bartek et al. >>2007<<). However, Lats2 is not activated in response to overt DNA damage (Aylon et al. 2006)." . _:b6279407 . _:b416968244 . _:b416968204 . _:b416968192 "2"^^ . . _:b6279406 . . _:b416968247 . _:b416968195 "2"^^ . _:b6279372 . _:b416968246 . _:b6279418 . _:b416968194 "2"^^ . _:b6279424 "Similarly, in astrocytoma, hypermethylation of the Lats2 promoter was detected in over 70% of the cases and associated with higher tumor grade and increased overall malignancy (Jiang et al. >>2006<<). Moreover, treatment of astrocytoma cell lines with a DNA methyltransferase inhibitor restored Lats2 levels and caused apoptosis, indicating that silencing of Lats2 is an important step in tumor cell resistance to apoptosis (Jiang et al." . _:b416968167 . _:b6279424 . . _:b416968251 . _:b416968241 . . _:b416968205 "2"^^ . _:b6279381 . _:b6279425 . _:b6279382 "results" . _:b416968240 . _:b416968204 "2"^^ . . _:b6279387 . _:b6279426 . _:b416968197 . _:b416968243 . _:b416968207 "2"^^ . _:b6279379 . _:b416968242 . _:b6279374 "Recently, we described a novel tumor suppressor axis, Lats2 (Large Tumor Suppressor 2)-Mdm2-p53, which is critical for the maintenance of proper chromosome number in the face of mitotic apparatus stress (Aylon et al. >>2006<<). In response to such stress, Lats2 protein departs the centrosomes, where it normally resides, and translocates to the nucleus." . _:b416968206 "2"^^ . . _:b416968253 . _:b416968201 "2"^^ . _:b416968252 . _:b416968200 "2"^^ . _:b416968203 "2"^^ . . _:b416968254 . _:b6279411 _:b6279416 . _:b416968202 "2"^^ . _:b6279411 _:b6279417 . _:b6279411 _:b6279418 . _:b6279411 _:b6279419 . _:b416968249 . . _:b6279411 _:b6279420 . _:b416968213 "2"^^ . _:b6279411 _:b6279421 . _:b6279411 _:b6279422 . _:b6279411 _:b6279423 . _:b416968248 . . _:b416968212 "2"^^ . . _:b416968251 . _:b6279411 _:b6279412 . _:b416968215 "2"^^ . _:b6279411 _:b6279413 . _:b6279411 _:b6279414 . _:b6279404 "Previously, Lats2 was found as a target of miR-372/3 in a screen for bypassing H-RasV12-induced, p53-dependent senescence in primary BJ cells (Voorhoeve et al. >>2006<<), suggesting that Lats2 may mediate the H-RasV12 signal that triggers p53 also in senescence." . . _:b6279411 _:b6279415 . _:b416968250 . _:b416968214 "2"^^ . _:b6279419 "Furthermore, by creating centrosome dysfunction (McDermott et al. 2006), prior loss of p16 might facilitate Lats2 activation through chronic mitotic apparatus stress (Aylon et al. >>2006<<). This may account for the occasional activation of Lats2 prior to H-RasV12 overexpression (Fig. 2B, upper left)." . _:b416968209 "2"^^ . . _:b416968208 "2"^^ . . _:b416968211 "2"^^ . _:b416968249 . _:b416968118 . _:b416968162 . . _:b416968210 "2"^^ . . _:b416968121 . _:b416968253 . _:b416968221 "2"^^ . _:b416968159 . _:b416968237 . _:b416968220 "2"^^ . . _:b416968223 "2"^^ . _:b6279384 . . _:b416968222 "2"^^ . _:b416968198 . _:b416968217 "2"^^ . _:b416968206 . _:b416968216 "2"^^ . _:b6279367 "For instance, deregulated Ras activity can induce cellular senescence, a process shown to depend at least in part on p53 and on the products of the INK4a tumor suppressor locus (Serrano >>1997<<). More recent evidence implicates Ras-induced p53-dependent senescence as a pivotal cancer-inhibitory mechanism also in animal models and in humans (Bartek et al. 2007; Ventura et al. 2007; Xue et al. 2007; Halazonetis et al. 2008)." . _:b416968219 "2"^^ . _:b416968173 . _:b416968218 "2"^^ . _:b416968229 "2"^^ . . . _:b416968228 "2"^^ . _:b6279423 . _:b416968231 "2"^^ . _:b6279412 "A number of studies suggest that oncogenic Ras can induce the expression of several DNA methyltransferases (Pruitt et al. >>2005<<; Chang et al. 2006), hence, it is possible that the silencing of Lats2 occurs only after H-RasV12 introduction." . _:b416968230 "2"^^ . _:b416968225 "2"^^ . _:b416968224 "2"^^ . _:b6279394 "It is also noteworthy that both these cell types lack functional p14ARF tumor suppressor protein (Burri et al. 2001; Milyavsky et al. >>2003<<), indicating that, at least in WI-38 and HCT116 cells, ARF is dispensable for the induction of Lats2 by oncogenic Ras." . _:b416968227 "2"^^ . . _:b416968226 "2"^^ . _:b416968158 . _:b416968237 "2"^^ . _:b6279402 "Like apoptosis, senescence serves as a barrier to prevent oncogene-driven cellular transformation (Lowe 1999; Bartkova et al. >>2006<<; Kim et al. 2007)." . _:b416968148 . _:b416968236 "2"^^ . . _:b416968239 "2"^^ . . _:b416968238 "2"^^ . _:b416968196 . _:b416968233 "2"^^ . _:b416968136 . _:b416968232 "2"^^ . _:b416968252 . _:b6279426 . _:b416968178 . . . . . _:b416968235 "2"^^ . _:b416968219 . . . _:b416968234 "2"^^ . . _:b416968245 "2"^^ . _:b6279416 "Notably, loss of p16(INK4A) expression, which took place in the course of WI-38/hTERTfast cell propagation in culture (Milyavsky et al. >>2003<<), may have accelerated DNA methylation (Reynolds et al. 2006), including that of the Lats2 promoter, thus generating a larger pool of cells to select from in response to H-RasV12-induced stress." . _:b416968172 . _:b416968144 . _:b416968244 "2"^^ . . . _:b416968247 "2"^^ .