Dissecting the Unique Role of the Retinoblastoma Tumor Suppressor during Cellular Senescence

May combine two claims.

Genes upregulated by RB loss often contain E2F binding sites and are highly expressed in growing but not quiescent or senescent cells.

“these genes often contained E2F binding sites and were highly expressed in growing but not in quiescent or senescent cells”

May combine two claims.

RB is uniquely needed to repress DNA replication E2F target genes during senescence, but not in other growth states.

“is uniquely required to repress these genes during senescence but not other growth states”

May combine two claims.

No significant RB peaks were observed outside the promoter region or in control precipitation.

“no significant peaks were observed outside the promoter region or in the control (beads-only) precipitation”

May combine two claims.

p130 binds the MCM5 promoter with 1.8-fold higher intensity in quiescent cells lacking RB, while its binding in senescent cells is unaffected by RB suppression.

“p130 bound the MCM5 gene promoter with a 1.8 Fold higher intensity in quiescent cells lacking RB than those expressing RB, while the binding of p130 in senescent cells was not affected by RB suppression”

May combine two claims.

RB loss significantly upregulates 741 genes in senescent cells, 120 in low-serum quiescent cells, 784 in confluent cells, and 291 in growing cells.

“we grouped genes that were significantly upregulated by RB loss in senescent, low serum, confluent and growing conditions (741, 120, 784, and 291 respectively; Table S2)”

May combine two claims.

SASP genes are still induced when each RB family member is individually suppressed and sometimes increase further without RB.

“these genes, some of which contribute to senescence, were still induced in the absence of each RB family member and, in some cases, their expression further increased in the absence of RB”

May combine two claims.

About 80% of RB peaks map within 5 kb upstream of known RefSeq transcriptional start sites, and about half are near promoters.

“About 80% of the peaks can be mapped within 5kb upstream of predicted transcriptional start sites of known refseq genes and about half are located near promoters”

May combine two claims.

Cluster C3 is enriched for DNA replication factors, especially pre-replication complex components, while cluster C4 is enriched for mitotic cell cycle factors.

“Gene ontology analysis revealed that genes in cluster C3 (Table S3; Replication cluster) were enriched for DNA replication factors (P<3.0×10−29 with Benjamini correction), particularly components of the pre-replication complex, while genes in cluster C4 (Table S3; Cell cycle cluster) were enriched for mitotic cell cycle factors (P<1.0×10−21 with Benjamini correction).”

May combine two claims.

Only RB knockdown, not p107 or p130 knockdown, lowers SA-β-gal accumulation in cells triggered to senesce.

“only those targeting RB reduced SA-β-gal accumulation (Figure 1C) and SAHF formation (Figure 1D and 1E) in cells triggered to senesce”

May combine two claims.

RB specifically binds the MCM3 promoter, an E2F target gene that is de-repressed without RB.

“we detected RB specifically bound to the promoter region of the MCM3 (Figure 3A), an E2F target genes de-repressed in the absence of RB”

May combine two claims.

p130 binding to many promoters increases in quiescent cells lacking RB, but no such effect occurs in senescent cells.

“whereas binding of p130 to many promoters increased in quiescent cells lacking RB, no effect was observed in senescent cells”

May combine two claims.

ChIP-seq identified 4525, 4214, and 8336 RB binding sites in growing, quiescent, and senescent cells, with 1281 overlapping peaks.

“We identified 4525, 4214 and 8336 RB binding sites in growing, quiescent and senescent cells, respectively, with an overlap of 1281 peaks”

May combine two claims.

Only RB knockdown, not p107 or p130 knockdown, reduces SAHF formation in cells triggered to senesce.

“only those targeting RB reduced SA-β-gal accumulation (Figure 1C) and SAHF formation (Figure 1D and 1E) in cells triggered to senesce”

May combine two claims.

Cluster C3 genes are highly expressed in RB-deficient senescent cells, whereas cluster C4 genes remain repressed.

“Whereas the genes in clusters C3 (Figure 2D) were highly expressed in RB-deficient senescent cells, the genes in cluster C4 (Figure 2E) remained repressed.”

May combine two claims.

RB shows about 3-fold higher affinity for cluster C3 genes and about 1.7-fold higher affinity for DNA replication factor genes than for other targets under all three conditions.

“RB showed an average 3-fold (P<1E-9) or 1.7-fold (p <1E-6) higher affinity for the genes in cluster C3 or “DNA replication factor” genes, respectively, under all three conditions”

May combine two claims.

Most RB ChIP-seq peaks are centered on the E2F motif, and RB binding intensity correlates with E2F motif quality.

“the majority of RB ChIP-seq peaks were centered at the E2F motif and that RB binding intensity correlated with the quality of E2F motif”

May combine two claims.

RB-deficient cells triggered to senesce show increased p53 serine 15 phosphorylation and increased p21.

“we noted an increase in p53 ser15 phosphorylation (Figure S5A) and a concomitant increase on its target gene p21 in RB-deficient cells triggered to senesce (Figure 7A)”

• AssumptionRests on an assumption: Increased p53 serine 15 phosphorylation indicates p53 activation.

May combine two claims.

Reintroducing wild-type or kinase-deficient cyclin E1 rescues the phenotype.

“we rescued the phenotype by reintroducing a shRNA-resistant cDNAs encoding either wild type or a kinase-deficient cyclin E1 (cyclin E1 KD-E)”

May combine two claims.

Co-expression of shRNAs against RB and p53, or p16 and p21, bypasses senescence and is linked to sustained expression of cell cycle/mitotic E2F targets (cyclin A, B, cdc2), though to different extents.

“Similarly, cells co-expressing shRNAs targeting RB and p53, or p16 and p21, bypass senescence (data not shown), which is associated with sustained expression of cell cycle/mitotic E2F targets (e.g.cyclin A, B, cdc2), albeit to varying degrees (Figure 7E).”

May combine two claims.

Suppression of cyclin E1 reduces MCM2-positive cells, indicating inefficient MCM2 loading onto chromatin.

“suppression of cyclin E1 caused a significant reduction in MCM2-positive cells (Figure 6C), indicating inefficient loading of MCM2 onto chromatin”

Probably not a substantial claim: may not pass S3 (load-bearing).

Cells co-suppressing p16 and p21 do not hyperinduce cyclin E.

“Interestingly, cells co-suppressing p16 and p21 did not hyperinduce cyclin E, perhaps owing to incomplete knockdown or a subtly different mechanism of senescence escape.”

May combine two claims.

Only a fraction of RB target genes are de-repressed in the absence of RB, and only in senescence.

“Still, only a fraction of RB target genes are de-repressed in the absence of RB and only in senescence.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Human vulvar intraepithelial neoplasias (VIN) that bypassed senescence via inactivation of RB family and p53 (Santegoets et al., 2007) also show hyperinduction of the replication factors studied here (Figure S1E).

“It remains to be determined whether our observations in fibroblasts can be extended to other cells types, though it is interesting that, human vulvar intraepithelial neoplesias (VIN) that have bypassed senescence through inactivation of the RB family and p53 (Santegoets et al., 2007) also show hyperinduction of the replication factors studied here (Figure S1E).”

• cites Santegoets et al., 2007 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Our analyses show that the primary targets of RB-mediated repression are genes with E2F binding sites, and RB preferentially associates with a subset of E2F targets.

“By identifying thousands of RB binding sites throughout the genome, our analyses decisively show that the primary targets of RB mediated repression are genes harboring E2F binding sites and, moreover, reveal that RB preferentially associates with a subset of E2F targets.”

May combine two claims.

Cyclin E was a key RB target in senescence, expressed at low levels in growing cells but strongly upregulated in RB-suppressed senescent cells.

“One key RB target in senescence was cyclin E, which was expressed at low levels in growing cells but strongly upregulated in RB-suppressed cells triggered to senesce.”

May combine two claims.

In human cells, RB is dispensable for cell cycle exit into quiescence; any RB family member can repress E2F target genes in this setting.

“In human cells, we see that RB is dispensable for cell cycle exit into quiescence – instead, any RB family member could repress E2F target genes in this setting [see also (Rayman et al., 2002)];”

• cites Rayman et al., 2002 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Inappropriate cyclin E1 expression in RB-deficient cells causes sustained MCM loading onto chromatin in a CDK-independent manner, unscheduled DNA replication, and endoreplication.

“Similarly, we see that the inappropriate expression of cyclin E1 in RB-deficient cells leads to sustained loading of MCM proteins onto chromatin in a CDK-independent manner, unscheduled DNA replication, and endoreplication.”

May combine two claims.

Although RB is needed for some senescence features, most gene expression changes in senescence are RB-independent.

“Thus, while RB is required for some aspects of the senescence program, the vast majority of changes are RB-independent.”

May combine two claims.

Each shRNA efficiently and specifically reduces its intended RB family protein.

“each shRNA efficiently and specifically represses its target RB family protein”

May combine two claims.

The ChIP-seq experiments were reproducible, with high overlap of top RB binding sites in biological replicates.

“the ChIP-seq experiments were reproducible (Pearson correlation coefficient 0.84) (Figure S2B), and about 89.7% and 76.5% of the top 1000 and top 2000 RB binding sites were identified in biological replicates, respectively”

May combine two claims.

Loss of RB shifts the equilibrium toward p130, which may explain why individual RB protein loss does not affect E2F target expression in quiescent cells.

“Loss of RB shifts the equilibrium towards p130 perhaps explaining why loss of individual RB proteins does not effect expression of E2F targets in quiescent cells.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Somatic mutations in p107 or p130 are rare in human cancers.

“somatic mutations affecting p107 or p130 are rare in human cancers (Burkhart and Sage, 2008)”

• cites Burkhart and Sage, 2008 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Without this checkpoint, proliferation continues unchecked, explaining how RB and p53 loss cooperate to bypass senescence.

“In the absence of this checkpoint, proliferation continues unabated, thus providing an explanation for how RB and p53 loss cooperate to bypass senescence.”

May combine two claims.

p21 loss reveals the full capacity of RB-deficient cells to aberrantly replicate DNA, explaining how RB and p53 cooperate to promote senescence.

“Regardless, p21 loss reveals the full capacity of RB deficient cells to aberrantly replicate DNA, thus explaining how RB and p53 cooperate to promote senescence.”

Classified as probably not a substantial claim.

Loss-of-function mutations in RB or its signaling network are considered necessary for cancer development.

“Loss-of-function mutations in the retinoblastoma gene product (RB) or its signaling network are considered requisite for cancer development”

• cites Burkhart and Sage, 2008 sourceNot checked yet

  Not checked in the free run.

• cites Rowland and Bernards, 2006Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

RB negatively regulates the E2F family of transcription factors at the G1-S transition.

“The best-characterized RB activity relates to its ability to control the G1-S transition, where it negatively regulates the E2F family of transcription factors.”

May combine two claims.

RB has a non-redundant role in binding and repressing E2F target genes involved in DNA replication, especially in senescent cells, reflecting a unique activity of RB in repressing DNA replication as cells exit into senescence.

“By performing a series of genome wide analyses, we see that RB has a non-redundant role in binding and repressing E2F target genes that are directly involved in DNA replication, particularly in senescent cells.”

• Also stated in: § RB action in tumor suppression · plain

  “Our results reveal a unique and non-redundant role for RB in preventing DNA replication in cells undergoing senescence.”

• Also stated in: § Chip-seq analysis · boosted

  “By coupling RNAi technology with a genome-wide analysis of gene expression and RB chromatin binding, we identified a unique and specific activity of RB in repressing DNA replication as cells exit the cell cycle into senescence.”

May combine two claims.

Spontaneous loss of p16 by DNA methylation in HMECs does not completely bypass senescence but is associated with polyploid cell accumulation (Romanov et al., 2001).

“and spontaneous loss of p16 due to DNA methylation in HMECs does not completely bypass senescence but is associated with the accumulation of polyploidy cells (Romanov et al., 2001).”

• cites Romanov et al., 2001 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

A similar situation has been observed for androgen receptor studies and may suggest compensation or redundancy in transcriptional control (Massie et al., 2007).

“Such a situation has also been observed for similar studies on the androgen receptor and may suggest compensation or redundancy in transcriptional control (Massie et al., 2007).”

• cites Massie et al., 2007 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

RB is dispensable for many gene expression changes in senescence but uniquely and specifically represses a subset of E2F target genes required for DNA replication.

“Here we see that RB is dispensable for many of the gene expression changes that accompany senescence but, instead, uniquely and specifically represses a subset of E2F target genes required for DNA replication.”

May combine two claims.

p53 promotes senescence by transactivating growth inhibitory genes, while RB represses growth-promoting genes, sometimes via heterochromatization of E2F target genes (Narita et al., 2003; Zhang et al., 2005).

“Whereas p53 promotes senescence by transactivating growth inhibitory genes, RB represses growth-promoting genes, which, in some cases, may involve heterochromatization of E2F target genes (Narita et al., 2003; Zhang et al., 2005).”

• cites Narita et al., 2003 sourceNot checked yet

  Not checked in the free run.

• cites Zhang et al., 2005 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

In cells triggered to senesce, RB loss causes sustained pre-replication complex loading, aberrant DNA replication, and, without a second checkpoint, proliferation and genomic instability.

“Consequently, in cells triggered to senesce, RB loss leads to sustained loading of pre-replication complexes, aberrant DNA replication and – in the absence of a second cell cycle checkpoint – proliferation and genomic instability.”

Classified as probably not a substantial claim. May combine two claims.

The senescence program acts as a general anti-proliferative stress response and is considered a potent tumor suppressive mechanism in vivo.

“the senescence program acts as a general anti-proliferative stress response and is considered a potent tumor suppressive mechanism in vivo [reviewed in (Narita and Lowe, 2005) (Prieur and Peeper, 2008)]”

• cites Narita and Lowe, 2005Not checked yet

  Not checked in the free run.

• cites Prieur and Peeper, 2008 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Previous studies suggested that RB family proteins regulate different E2F targets (Hurford et al., 1997) and that RB loss increases replication complex loading on chromatin (Srinivasan et al., 2007).

“Although previous studies hinted that RB family proteins can regulate different E2F targets (Hurford et al., 1997) and that loss of RB can ultimately lead to increased loading of replications complexes on chromatin (Srinivasan et al., 2007), the selectivity of RB towards repressing replication genes was not appreciated.”

• cites Hurford et al., 1997 sourceNot checked yet

  Not checked in the free run.

• cites Srinivasan et al., 2007 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

RB is required to halt DNA synthesis during exit into senescence, and its loss causes unscheduled DNA replication and sometimes endoreplication.

“these results demonstrate that RB is required to halt DNA synthesis during cell cycle exit into senescence, such that cells lacking RB undergo unscheduled DNA replication and, in some cases, endoreplication”

May combine two claims.

Cells lacking RB eventually arrest by engaging a second proliferation barrier mediated by p21; RB loss triggers a p53/p21-dependent checkpoint that prevents escape from senescence.

“Consistent with a role for p53 in senescence, cells lacking RB eventually arrest because they engage a second proliferation barrier mediated by the p21 cyclin-dependent kinase inhibitor.”

• Also stated in: § Chip-seq analysis · plain

  “RB loss triggers a p53/p21 dependent checkpoint that prevents escape from senescence”

May combine two claims.

RB inactivation prevents SAHF accumulation and cooperates with p53 loss to bypass senescence in human diploid fibroblasts.

“prevents SAHF accumulation and cooperates with p53 loss to bypass senescence in human diploid fibroblasts (Narita et al., 2003; Voorhoeve and Agami, 2003)”

• cites Narita et al., 2003 sourceNot checked yet

  Not checked in the free run.

• cites Voorhoeve and Agami, 2003Not checked yet

  Not checked in the free run.

May combine two claims.

The RB proteins have redundant and/or compensatory functions.

“There is ample evidence that the RB proteins have redundant and/or compensatory functions (Sage et al., 2000).”

• cites Sage et al., 2000 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Assembly of the pre-RC on origins is a crucial and limiting step in DNA synthesis that occurs once per cell cycle before S phase.

“Assembly of the pre-RC on origins of replication is a crucial and limiting step in the synthesis of DNA occurring only once per cell cycle and during a short window of time prior to S phase (Stillman, 1996).”

• cites Stillman, 1996 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

RB's importance as a tumor suppressor is evident from frequent inactivating mutations in RB or its pathway in human cancer; the RB pathway is thought to be disabled in virtually all tumor cells (Burkhart and Sage, 2008).

“The importance of RB as a tumor suppressor gene is evident by the frequency of inactivating mutations that target RB or its pathway in human cancer; indeed, the RB pathway is thought to be disabled in virtually all tumor cells (Burkhart and Sage, 2008).”

• cites Burkhart and Sage, 2008 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

When RB is lost, cells undergo inappropriate DNA replication, aberrant proliferation, and genomic instability-processes exacerbated by other checkpoint defects and contributing to malignant tumors.

“Hence, when RB is lost, cells undergo inappropriate DNA replication, aberrant proliferation, and display genomic instability – processes that are exacerbated by other checkpoint defects and contribute to the development of malignant tumors.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Cyclin E1 is a bona fide oncogene overexpressed in many human tumor types and often associated with poor prognosis (Butt et al., 2008).

“In this regard, it is interesting that a key target of RB-mediated repression in senescence is cyclin E1 – a bona fide oncogene whose over-expression occurs in many human tumor types and is often associated with poor prognosis (Butt et al., 2008).”

• cites Butt et al., 2008 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

These results link the kinase-independent function of cyclin E1, MCM complex loading onto chromatin, and endoreplication, and show how RB coordinately shuts down DNA synthesis as cells enter senescence.

“These results establish a connection between the kinase-independent function of cyclin E1, loading of MCM complexes onto chromatin, and endoreplication, and highlight how RB acts in a coordinated way to shut down DNA synthesis as cells enter senescence.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Cyclin E promotes cell cycle progression by activating CDKs (Zhang, 2007).

“Since cyclin E promotes cell cycle progression by activating CDKs (Zhang, 2007), its selective control by RB stands out from other RB targets directly involved in DNA replication.”

• cites Zhang, 2007 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The discrepancy in our results may relate to cell type or species differences or to HES1 status, which may determine quiescence reversibility (Sang et al., 2008).

“The apparent discrepancy in our results may relate to cell type or species differences or the status of the helix-loop-helix protein HES1, which may play a crucial role in determining the reversibility of the quiescent state (Sang et al., 2008).”

• cites Sang et al., 2008 sourceNot checked yet

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