p53 Deficiency Rescues the Adverse Effects of Telomere Loss and Cooperates with Telomere Dysfunction to Accelerate Carcinogenesis

Loss of telomere function and p53 deficiency cooperated to initiate the transformation process.

“Correspondingly, the loss of telomere function and p53 deficiency cooperated to initiate the transformation process.”

Coincident with severe telomere shortening and associated genomic instability, p53 is activated, leading to growth arrest and/or apoptosis.

“Coincident with severe telomere shortening and associated genomic instability, p53 is activated, leading to growth arrest and/or apoptosis.”

CAT activity was strictly dependent upon p53.

“This activity was strictly dependent upon p53, since p53−/− and G6 mTR−/− p53−/− MEF cultures showed only background levels of CAT activity.”

p53 mediates the adverse effects of critically shortened telomeres.

“We found that p53 mediates the adverse effects of critically shortened telomeres.”

DNA content analyses of G6 mTR−/− p53+/+ MEFs revealed a profile consistent with a biphasic block as evidenced by marked accumulation of cells in G1 and G2/M phases.

“As shown in Figure 3A, DNA content analyses of G6 mTR−/− p53+/+ MEFs revealed a profile consistent with a biphasic block as evidenced by marked accumulation of cells in G1 and G2/M phases.”

Analysis of metaphase spreads derived from G6 mTR−/− p53+/+ MEFs showed a rate of 0.42 (SD 0.71) chromosomal fusions per metaphase.

“Indeed, analysis of metaphase spreads derived from G6 mTR−/− p53+/+ MEFs showed a rate of 0.42 (SD 0.71) chromosomal fusions per metaphase, a rate consistent with previous studies (Figure 3D) (Blasco et al. 1997; Greenberg et al. 1999; Rudolph et al. 1999”

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p53-independent cell death becomes a more consistent feature in G7 and G8, since all of the five G7-G8 mTR−/− p53−/− testis samples showed significant germ cell depletion microscopically.

“Furthermore, p53-independent cell death becomes a more consistent feature in G7 and G8, since all of the five G7–G8 mTR−/− p53−/− testis samples showed significant germ cell depletion microscopically (Figure 2D).”

A relative increase in p53−/− mice was evident in G7 and G8 mTR−/− background (24% and 38%, respectively).

“However, a relative increase in p53−/− mice was evident in G7 and G8 mTR−/− background (24% and 38%, respectively).”

Gross testis size was consistently rescued to near normal in G6 mTR−/− p53−/− mice compared to G6 mTR−/− p53+/+ littermates.

“Gross testis size was consistently rescued to near normal in G6 mTR−/− p53−/− mice compared to G6 mTR−/− p53+/+ littermates (see Figure 2A for representative comparison).”

Loss of telomere function and p53 deficiency can cooperate to enhance transformation.

“Furthermore, loss of telomere function and p53 deficiency can cooperate to enhance transformation.”

Transfection of the mTR genomic fragment restored telomerase activity in mTR−/− cells.

“Transfection of this mTR genomic fragment restored telomerase activity in mTR−/− cells (Figure 4B).”

Modest attenuation of the cell cycle block was evident in G6 mTR−/− p53+/− cultures, suggesting functional haploinsufficiency.

“Modest attenuation of the cell cycle block was evident in the G6 mTR−/− p53+/− cultures, suggesting functional haploinsufficiency.”

Loss of p53 does not appear to eliminate the cell cycle blocks completely, since G6 mTR−/− p53−/− MEFs still exhibited fewer cells in S phase (33% vs. 55%) and more cells in G2/M (14% vs. 7%) than mTR+/+ p53−/− MEFs.

“However, loss of p53 does not appear to eliminate the cell cycle blocks completely, since G6 mTR−/− p53−/− MEFs still exhibited fewer cells in S phase (33% vs. 55%) and more cells in G2/M (14% vs. 7%) than mTR+/+ p53−/− MEFs.”

Similar results were obtained for primary thymocytes, irrespective of p53 status.

“Similar results were obtained for primary thymocytes, irrespective of p53 status (Figure 3C).”

Endogenous p21CIP1/WAF1 levels were increased 3- to 4-fold in late-generation G6 mTR−/− p53+/+ MEFs compared to early-generation G2 mTR−/− p53+/+ MEFs.

“Endogenous p21CIP1/WAF1 levels were increased 3- to 4-fold in late-generation G6 mTR−/− p53+/+ MEFs compared to early-generation G2 mTR−/− p53+/+ MEFs (Figure 1C).”

Most of the G5-G7 mTR−/− p53−/− mice showed improved histology compared to their age-matched controls with competent p53 function.

“Most of the G5–G7 mTR−/− p53−/− mice showed improved histology compared to their age-matched controls with competent p53 function.”

In mTR−/− mice, a decrease in testis size that correlated with germ cell depletion by histology was first evident in G5 mTR−/− animals and became more prevalent in G6 mTR−/− mice.

“In mTR−/− mice, a decrease in testis size that correlated with germ cell depletion by histology was first evident in G5 mTR−/− animals and became more prevalent in G6 mTR−/− mice (Lee et al. 1998”

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When established cell lines were injected into SCID mice, mTR lines and vector lines formed tumors with similar efficiency.

“However, when these established cell lines were injected into SCID mice, mTR lines and vector lines formed tumors with similar efficiency (Figure 4D).”

In G6 mTR−/− p53+/− intercrosses, an unusually high rate of productive mating pairs (11 of 22) was observed compared to G6 mTR−/− p53+/+ matings.

“In the G6 mTR−/− p53+/− intercrosses, we observed an unusually high rate of productive mating pairs (11 of 22 mating pairs) compared to G6 mTR−/− p53+/+ matings (Lee et al. 1998”

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Addition of mTR consistently suppressed the rate of Myc/RAS focus formation in G6 mTR−/− p53−/− MEFs.

“we demonstrated that the addition of mTR consistently suppressed the rate of Myc/RAS focus formation in G6 mTR−/− p53−/− MEFs (Table 2)”

MEFs from mTR+/+ mice and from late-generation mTR−/− mice exhibited marked differences in the proportion of cells expressing p53.

“MEFs from mTR+/+ mice and from late-generation mTR−/− mice exhibited marked differences in the proportion of cells expressing p53 as determined by immunohistochemistry.”

The G8 mTR−/− p53−/− mouse that exhibited relatively normal histology had significantly longer telomere length than the other G8 mTR−/− p53−/− mouse.

“Of note, the G8 mTR−/− p53−/− mouse that exhibited a relatively normal histology (1+ germ cell depletion, Table 1) had significantly longer telomere length by flow FISH determination (data not shown) than the other G8 mTR−/− p53−/− mouse.”

CAT activity was increased approximately 2-fold in G6 mTR−/− MEFs compared to mTR+/+ p53+/+ controls at identical passage number.

“After normalization for transfection efficiency, CAT activity was increased approximately 2-fold in G6 mTR−/− MEFs compared to mTR+/+ p53+/+ controls at identical passage number (Figure 1D).”

TUNEL assays demonstrated that the restoration of germ cells in the G5-G7 mTR−/− p53−/− testis is accompanied by a dramatic reduction of apoptosis to levels observed in mTR+/+ testis.

“TUNEL assays demonstrated that the restoration of germ cells in the G5–G7 mTR−/− p53−/− testis is accompanied by a dramatic reduction of apoptosis to levels observed in mTR+/+ testis (Figure 2C).”

p21CIP1/WAF1 induction in mTR−/− cells is largely p53 dependent.

“As anticipated, p21CIP1/WAF1 was not detected in G6 mTR−/− p53−/− cells, indicating that p21CIP1/WAF1 induction in mTR−/− cells is largely p53 dependent.”

The average weight of mTR tumors was not significantly different from that of vector tumors.

“Furthermore, the average weight of mTR tumors was not significantly different from that of the vector tumors.”

Restoration of telomerase activity in mTR+/+ p53−/− MEFs had no significant effect on focus formation.

“while restoration of telomerase activity in mTR+/+ p53−/− MEFs had no significant effect (data not shown).”

All G6 mTR−/− MEF cultures regardless of p53 status exhibited an equivalent and marked decline in mean telomere signal relative to mTR+/+ MEFs (75% reduction).

“As shown in Figure 3B, all of the G6 mTR−/− MEF cultures regardless of p53 status exhibited an equivalent and marked decline in mean telomere signal relative to mTR+/+ MEFs (75% reduction).”

p53−/− mice were underrepresented as a percentage of total mice in late-generation mTR−/− background (19%, 15%, and 14% for G4, G5, and G6 respectively).

“p53−/− mice were underrepresented as a percentage of total mice in late-generation mTR−/− background (19%, 15%, and 14% for G4, G5, and G6 respectively) (Figure 1E).”

Myc/RAS foci transfected with mTR exhibited a significant enhancement in subcloning efficiency: 23 of 26 (89%) mTR foci were established as permanent cell lines compared with 19 of 32 (59%) vector foci (p = 0.018).

“Myc/RAS foci transfected with mTR exhibited a significant enhancement in subcloning efficiency—23 of 26 (89%) mTR foci were successfully established as permanent cell lines compared with only 19 of 32 (59%) vector foci (Figures 4C, p value = 0.018).”

As telomere function worsens in later generations of mTR deficiency, germ cell death by p53-independent mechanisms becomes evident.

“As telomere function worsens in later generations of mTR deficiency, germ cell death by p53-independent mechanisms becomes evident.”

Productive matings arising from G7 mTR−/− p53+/− pairs (5 of 17) yielded G8 mTR−/− offspring of all three p53 genotypes.

“Significantly, we also observed productive matings arising from G7 mTR−/− p53+/− pairs (5 of 17 mating pairs) to yield G8 mTR−/− offspring of all three p53 genotypes.”

Telomere shortening in the absence of telomerase plays a critical role in signaling entry into senescence.

“Thus, telomere shortening in the absence of telomerase plays a critical role in signaling entry into senescence”

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• cites Counter et al. 1998 sourceNot checked yet

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The oncogenic potential of a would-be cancer cell bearing short telomeres may depend upon its genetic profile.

“These results indicate that the oncogenic potential of a would-be cancer cell bearing short telomeres may depend upon its genetic profile.”

SV40 large T antigen efficiently transformed cells from late-generation mTR−/− mice despite evidence for telomere dysfunction.

“In contrast, SV40 large T antigen efficiently transformed cells from late-generation mTR−/− mice despite evidence for telomere dysfunction (Blasco et al. 1997; Greenberg et al. 1999”

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p53 plays a key role in the cellular response to telomere dysfunction in both normal and neoplastic cells.

“Together, these studies establish a key role for p53 in the cellular response to telomere dysfunction in both normal and neoplastic cells”

There is a biologically relevant stage of advanced crisis, termed genetic catastrophe.

“identify a biologically relevant stage of advanced crisis, termed genetic catastrophe.”

The inactivation of Rb and/or p53 may account for the efficient transformation of cells with significant telomere loss by SV40 large T antigen.

“The inactivation of Rb and/or p53 may account for the efficient transformation of cells with significant telomere loss by SV40 large T antigen.”

In cancer-prone mice rendered doubly null for the INK4a tumor suppressor and mTR, shortened telomeres markedly reduced both cancer incidence and transformation by cellular oncogenes.

“In cancer-prone mice rendered doubly null for the INK4a tumor suppressor and mTR, shortened telomeres markedly reduced both cancer incidence and transformation by cellular oncogenes (Greenberg et al., 1999”

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Telomerase provides a significant advantage in steps following the initiation of cellular transformation.

“Taken together, the differences in the subcloning efficiency of the mTR versus vector cells and their similarity in tumorigenic potential following prolonged passage in culture argue that telomerase provides a significant advantage in steps following the initiation of cellular transformation.”

Once telomerase-deficient cells have adapted to existing telomere dysfunction, they grow as efficiently as telomerase-expressing cells.

“However, once telomerase-deficient cells have adapted to the existing telomere dysfunction, they grow as efficiently as telomerase-expressing cells.”

The specific response to telomere dysfunction can be either growth arrest or apoptosis depending upon cell type, p53 pathway status, and physiological context.

“Similar to well-characterized DNA damage checkpoints, the specific response to telomere dysfunction can be either growth arrest or apoptosis depending upon cell type, p53 pathway status, and physiological context.”

Germ cell apoptosis coincident with telomere loss is mediated by p53.

“These findings indicate that germ cell apoptosis coincident with telomere loss is mediated by p53.”

Loss of telomere function in late-generation (G4 onward) mTR−/− mice adversely affects the morphology and physiology of diverse cell types and organ systems.

“Loss of telomere function in late-generation (G4 onward) mTR−/− mice has been shown to adversely affect the morphology and physiology of diverse cell types and organ systems (Lee et al. 1998; Rudolph et al. 1999”

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Cell death in the context of extreme telomere dysfunction, genetic instability, and p53 deficiency in G7 and G8 mTR−/− mice may represent a more accurate model of the late stage of crisis experienced by human cells that bypass senescence.

“Therefore, cell death in the context of extreme telomere dysfunction, genetic instability, and p53 deficiency in G7 and G8 mTR−/− mice may represent a more accurate model of the late stage of crisis experienced by human cells that bypass senescence (see Discussion).”

p53 plays a key role in the cellular response to telomere dysfunction in vivo in both normal and neoplastic cells, particularly during early stages of genomic crisis.

“Nevertheless, we have established that p53 plays a key role in the cellular response to telomere dysfunction in vivo in both normal and neoplastic cells, a role that is particularly prominent during the early stages of genomic crisis.”

• AssumptionRests on an assumption: The mTR−/− mouse is a valid model for studying the consequences of telomere loss in vivo.

Late crisis is not a checkpoint but represents the consequence of unimpeded proliferation in the absence of functional telomeres and p53.

“We propose that late crisis is not a checkpoint but instead represents the consequence of unimpeded proliferation in the absence of two powerful genome-stabilizing factors: functional telomeres and p53.”

• AssumptionRests on an assumption: Human cells require inactivation of Rb and/or p53 to bypass the major checkpoint that limits replicative lifespan: cellular senescence.
• AssumptionRests on an assumption: Rb/p53 inactivation allows continued cell division beyond senescence, progressive telomere shortening, and entry into crisis, from which only rare survivors emerge.

p53 is an important effector in a pathway activated by loss of telomere function.

“Together these data establish p53 as an important effector in a pathway activated by loss of telomere function.”

• AssumptionRests on an assumption: The mTR−/− mouse is a valid model for studying the consequences of telomere loss in vivo.

The rescue of proliferation, cellularity, and cell death in telomerase-deficient mice is due to loss of p53 checkpoint functions.

“The rescue of proliferation, cellularity, and cell death is therefore due to loss of p53 checkpoint functions.”

Critical telomere shortening and associated chromosomal instability correlate with an increase in the level and activation of the p53 protein.

“Together, these data indicate that critical telomere shortening and the associated chromosomal instability correlate with an increase in the level and the activation of the p53 protein.”

Cell death in early crisis is p53 dependent.

“Cell death in early crisis is p53 dependent.”

The p53-mediated pathway can dramatically affect the consequence of telomere shortening during carcinogenesis.

“Furthermore, this p53-mediated pathway can dramatically affect the consequence of telomere shortening during carcinogenesis.”

• AssumptionRests on an assumption: The mTR−/− mouse is a valid model for studying the consequences of telomere loss in vivo.

As telomere function worsens in G7 and G8, p53+/+ and p53+/− mice are at a significant survival disadvantage compared to p53−/− mice.

“These data indicate that as telomere function worsens in G7 and G8, p53+/+ and p53+/− mice are at a significant survival disadvantage compared to p53−/− mice.”

Addition of mTR to Myc/RAS cotransfections of late-generation mTR−/− INK4a−/− MEFs enhanced the rate of focus formation relative to empty vector controls.

“Most significantly, in contrast to late-generation mTR−/− p53−/− MEFs, addition of mTR to the Myc/RAS cotransfections of late-generation mTR−/− INK4a−/− MEFs enhanced the rate of focus formation relative to empty vector controls.”

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Activation of telomerase enhances cell survival possibly through avoidance of the p53-independent death of genetic catastrophe.

“Thus, the activation of telomerase enhances cell survival possibly through avoidance of the p53-independent death of genetic catastrophe (Figure 5).”

The period defined as crisis in cultured cells can be divided into two phases: early crisis and late crisis (genetic catastrophe).

“Our in vivo and cell culture data suggest that the period defined as crisis in cultured cells can be divided into two phases: “early crisis” and “late crisis,” the latter we term “genetic catastrophe” (Figure 5).”

• AssumptionRests on an assumption: Human cells require inactivation of Rb and/or p53 to bypass the major checkpoint that limits replicative lifespan: cellular senescence.
• AssumptionRests on an assumption: Rb/p53 inactivation allows continued cell division beyond senescence, progressive telomere shortening, and entry into crisis, from which only rare survivors emerge.

In other cells, increased aneuploidy or fusion-bridge-breakage may facilitate genetic changes that promote survival, such as oncogene activation, tumor suppressor deletion, reactivation of telomere maintenance mechanisms, or adaptation to telomere dysfunction.

“In other cells, increased aneuploidy or fusion-bridge-breakage may facilitate genetic changes that promote survival, such as oncogene activation, tumor suppressor deletion, reactivation of telomere maintenance mechanisms, or adaptation to telomere dysfunction (e.g., Myc/RAS-expressing G6 mTR−/− p53−/− MEFs) (Figure 5).”

The p53 pathway may alter the cellular response to telomere loss in both normal and neoplastic cells.

“The contrasting effect of telomere loss on cellular transformation phenotypes in cells that have intact or inactivated DNA damage pathways suggested that the p53 pathway may alter the cellular response to telomere loss in both normal and neoplastic cells.”

Cells that cannot reestablish telomere maintenance or tolerate genomic instability (e.g., germ cells in G7 and G8) will die.

“Cells that cannot reestablish telomere maintenance or tolerate genomic instability (e.g., germ cells in G7 and G8) will die.”

Telomere dysfunction cooperates with p53 deficiency to facilitate cellular transformation.

“This unanticipated result indicates that telomere dysfunction cooperates with p53 deficiency to facilitate cellular transformation.”

• AssumptionRests on an assumption: Focus formation reflects the earliest stages of oncogenic transformation.

The mTR−/− INK4a−/− study provides strong in vivo support for the hypothesis that maintenance of telomere function is required for efficient tumorigenesis.

“The mTR−/− INK4a−/− study provides strong in vivo support for the hypothesis that maintenance of telomere function is required for efficient tumorigenesis.”

In cancer cells harboring intact DNA damage response pathways such as INK4a−/− p53+/+ genotype, pharmacological inhibition of telomerase and subsequent telomere shortening would be predicted to be more therapeutically efficacious.

“However, in cancer cells harboring intact DNA damage response pathways such as those with an INK4a−/− p53+/+ genotype, pharmacological inhibition of telomerase and subsequent telomere shortening would be predicted to be more therapeutically efficacious.”