Reprogramming of replicative senescence in hepatocellular carcinoma-derived cells

Tumor cells have the capacity to proliferate indefinitely that is qualified as replicative immortality. This ability contrasts with the intrinsic control of the number of cell divisions in human somatic tissues by a mechanism called replicative senescence. Replicative immortality is acquired by inactivation of p53 and p16INK4a genes and reactivation of hTERT gene expression. It is unknown whether the cancer cell replicative immortality is reversible. Here, we show the spontaneous induction of replicative senescence in p53-and p16INK4a-deficient hepatocellular carcinoma cells. This phenomenon is characterized with hTERT repression, telomere shortening, senescence arrest, and tumor suppression. SIP1 gene (ZFHX1B) is partly responsible for replicative senescence, because short hairpin RNA-mediated SIP1 inactivation released hTERT repression and rescued clonal hepatocellular carcinoma cells from senescence arrest.     

Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes

Replicative senescence of human diploid fibroblasts (HDFs) or melanocytes is caused by the exhaustion of their proliferative potential. Stress-induced premature senescence (SIPS) occurs after many different sublethal stresses including H(2)O(2), hyperoxia, or tert-butylhydroperoxide. Cells in replicative senescence share common features with cells in SIPS: morphology, senescence-associated beta-galactosidase activity, cell cycle regulation, gene expression and telomere shortening. Telomere shortening is attributed to the accumulation of DNA single-strand breaks induced by oxidative damage. SIPS could be a mechanism of accumulation of senescent-like cells in vivo. Melanocytes exposed to sublethal doses of UVB undergo SIPS. Melanocytes from dark- and light- skinned populations display differences in their cell cycle regulation. Delayed SIPS occurs in melanocytes from light-skinned populations since a reduced association of p16(Ink-4a) with CDK4 and reduced phosphorylation of the retinoblastoma protein are observed. The role of reactive oxygen species in melanocyte SIPS is unclear. Both replicative senescence and SIPS are dependent on two major pathways. One is triggered by DNA damage, telomere damage and/or shortening and involves the activation of the p53 and p21(waf-1) proteins. The second pathway results in the accumulation of p16(Ink-4a) with the MAP kinase signalling pathway as possible intermediate. These data corroborate the thermodynamical theory of ageing, according to which the exposure of cells to sublethal stresses of various natures can trigger SIPS, with possible modulations of this process by bioenergetics.     

Cell cycle regulation in H(2)O(2)-induced premature senescence of human diploid fibroblasts and regulatory control exerted by the papilloma virus E6 and E7 proteins

Many biomarkers of replicative senescence appear in stress-induced premature senescence (SIPS) of human diploid fibroblasts (HDFs). The mRNA level of key cell cycle regulators was studied in H(2)O(2)-induced premature senescence of HDFs expressing or not the papillomavirus E6 and E7 proteins, which enhanced, respectively, the proteolysis of p53 and Rb. The CdKI's p21(waf-1) and p16(Ink-4a) were found overexpressed in H(2)O(2)-induced premature senescence, while p19(Ink-4d)and p27(Kip-1) were repressed. The results obtained in E6 HDFs suggest that p21(waf-1) and p16(Ink-4a) overexpressions are p53-independent, while p27(Kip-1) and p19(Ink-4d) down-regulations are p53-dependent.E6 regulated Rb, p130, p53 and p16(Ink-4a) mRNA level in non-stressing conditions, and regulated p130, p107, p53, p19(Ink-4d), p27(Kip-1) mRNA level in SIPS. SIPS modified the E6-mediated regulatory control on p107, p16(Ink-4a), p19(Ink-4d) and p27(Kip-1) mRNA level, when compared to normal conditions.E7 regulated the mRNA level of all the genes studied, in all conditions, suggesting that the Rb family or other E7-interacting proteins might modify the expression of these genes. SIPS modified strongly the E7-mediated regulatory control on p107, p16(Ink-4a), p19(Ink-4d), p27(Kip-1), p21(Waf-1) and Rb mRNA level, when compared to normal conditions. Further work is ongoing to test whether this E7-mediated regulatory control takes place through interactions with Rb or other E7-interacting proteins.     

Long-term cultured IL-2-dependent T cell lines demonstrate p16(INK4a) overexpression, normal pRb/p53, and upregulation of cyclins E or D2

Acquisition of an immortal phenotype by circumvention of the normal senescence program can be an important step in tumor development and progression. The regulation of life-span checkpoints is complex and abrogation of these processes can occur at different levels. To better understand these mechanisms in long-term cultured lymphocytes we have characterized two human long-term cultured IL-2-dependent T cell lines regarding telomere length, telomerase activity, and the expression of selected cell cycle regulators (pRb, p53, cyclin E, cyclin D1, cyclin D2, cyclin D3, cdk4, p16(INK4a), p21(WAF1), p27(KIP1), c-myc, bcl-2, and NPAT). We compared these cell lines with a primary T lymphoblast population with a limited life span from the same donor. Both T cell lines with extraordinary growth capacity showed telomere length stabilization, high telomerase activity and demonstrated wild-type pattern of pRb and p53 but strong p16(INK4a) protein expression. The growth inhibitory activity of p16(INK4a) seemed to be abrogated by enhanced expression of cyclin D2, cdk4, and c-myc in one T cell line and overexpression of cyclin E in the second T cell line.     

Angiotensin II-mediated oxidative DNA damage accelerates cellular senescence in cultured human vascular smooth muscle cells via telomere-dependent and independent pathways

Angiotensin II (Ang II) induces reactive oxygen species (ROS) production by human vascular smooth muscle cells (hVSMCs). ROS have been implicated in the development of both acute stress-induced premature senescence (SIPS) and chronic replicative senescence. Global oxidative DNA damage triggers SIPS and telomere DNA damage accelerates replicative senescence, both mediated via p53. This study tests the hypothesis that DNA is an important target for Ang II-induced ROS leading to senescence via telomere-dependent and independent pathways. DNA damage was quantified using the Comet assay, telomere DNA length by Southern blotting and hVSMC senescence by senescence-associated beta-galactosidase staining. Exposure to Ang II increased DNA damage in hVSMCs within 4 hours. Inhibition by an AT1 receptor antagonist (losartan metabolite: E3174) or catalase, confirmed that Ang II-induced DNA damage was AT1 receptor-mediated, via the induction of ROS. Acute exposure to Ang II resulted in SIPS within 24 hours that was prevented by coincubation with E3174 or catalase. SIPS was associated with increased p53 expression but was not dependent on telomere attrition because overexpression of human telomerase did not prevent Ang II-induced SIPS. Exposure to Ang II over several population doublings accelerated the rate of telomere attrition (by >2-fold) and induced premature replicative senescence of hVSMCs--an effect that was also attenuated by E3174 or catalase. These data demonstrate that Ang II-induced ROS-mediated DNA damage results in accelerated biological aging of hVSMCs via 2 mechanisms: (1) Acute SIPS, which is telomere independent, and (2) accelerated replicative senescence which is associated with accelerated telomere attrition.     

p53-dependent integration of telomere and growth factor deprivation signals

Ectopically expressed hTERT enables p16(INK4A)(-) human mammary epithelial cells to proliferate in the absence of growth factors, a finding that has led to the hypothesis that hTERT has growth regulatory properties independent of its role in telomere maintenance. We now show that telomerase can alter the growth properties of cells indirectly through its role in telomere maintenance, without altering growth stimulatory pathways. We find that telomere dysfunction, indicated by 53BP1/phosphorylated histone H2AX foci at chromosome ends, is present in robustly proliferating human mammary epithelial cells long before senescence. These foci correlate with increased levels of active p53. Ectopic expression of hTERT reduces the number of foci and the level of active p53, thereby decreasing sensitivity to growth factor depletion, which independently activates p53. The continuous presence of hTERT is not necessary for this effect, indicating that telomere maintenance, rather than the presence of the enzyme itself, is responsible for the increased ability to proliferate in the absence of growth factors. Our findings provide a previously unrecognized mechanistic explanation for the observation that ectopically expressed hTERT conveys growth advantages to cells, without having to postulate nontelomeric functions for the enzyme.     

Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor beta growth inhibition in p16INK4A(-) human mammary epithelial cells

Failures to arrest growth in response to senescence or transforming growth factor beta (TGF-beta) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16(INK4A) resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-beta. The ability to maintain growth in TGF-beta was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-beta resistance may explain our previously described gain of TGF-beta resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-beta resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.     

Distinct mechanisms of cell cycle arrest control the decision between differentiation and senescence in human neuroblastoma cells

Retinoic acid (RA) induces cell cycle arrest and differentiation of human neuroblastoma (NB) cells. Typically, NB cells differentiate along the neuronal lineage, but quiescent, "flat" cell types frequently have been described after treatment with differentiating agents. Two indistinguishable subclones of the cell line SK-N-SH, SK-N-SH-N (SH-N) and SK-N-SH-F (SH-F), display dramatically different responses to RA. In SH-N, RA induces neuronal differentiation, but in SH-F it transforms the small neuroblastic cells into large, flattened, epithelium-like cells. Here we analyze the mechanistic basis for the different effects of RA in the two NB subclones. First, we show that the flattened RA-treated SH-F expresses markers of cells undergoing replicative senescence. Inhibition of DNA synthesis by RA is significantly more rapid in SH-F than in SH-N. SH-F, which expresses basal amounts of p16(INK4A), responds to RA with elevation of p18(INK4C), marked down-regulation of cyclin D1, and swift inhibition of cyclin D-dependent kinases (cdks). Conversely, after addition of RA, SH-N retains cell cycling due to high expression of cyclin D1, the absence of Ink4 inhibitors, and accumulation of p21(Cip1). These changes result in sustained cdk activity. Accordingly, overexpression of p21(Cip1) but not p16(INK4A) induces neuronal differentiation of untreated NB cells. We propose that rapid inhibition of cdks by RA in NB leads to early cell cycle arrest, prevents neuronal differentiation, and results in a senescence-like state.     

Distinct and nonoverlapping roles for pRB and cyclin D:cyclin-dependent kinases 4/6 activity in melanocyte survival

Deregulation of the p16INK4a-cyclin D:cyclin-dependent kinases (cdk) 4/6-retinoblastoma (pRB) pathway is a common paradigm in the oncogenic transformation of human cells and suggests that this pathway functions linearly in malignant transformation. However, it is not understood why p16INK4a and cyclin D:cdk4/6 mutations are disproportionately more common than the rare genetic event of RB inactivation in human malignancies such as melanoma. To better understand how these complexes contribute to altered tissue homeostasis, we blocked cdk4/6 activation and acutely inactivated Rb by conditional mutagenesis during mouse hair follicle cycling. Inhibition of cdk4/6 in the skin by subcutaneous administration of a membrane-transducible TAT-p16INK4a protein completely blocked hair follicle growth and differentiation. In contrast, acute disruption of Rb in the skin of homozygous RbLoxP/LoxP mice via subcutaneous administration of TAT-Cre recombinase failed to affect hair growth. However, loss of Rb resulted in severe depigmentation of hair follicles. Further analysis of follicular melanocytes in vivo and in primary cell culture demonstrated that pRB plays a cell-autonomous role in melanocyte survival. Moreover, functional inactivation of all three Rb family members (Rb, p107, and p130) in primary melanocytes by treatment with a transducible TAT-E1A protein did not rescue the apoptotic phenotype. These findings suggest that deregulated cyclin D:cdk4/6 complexes and pRB perform nonoverlapping functions in vivo and provide a cellular mechanism that accounts for the low incidence of RB inactivation in cancers such as melanoma.     

Senescence-like changes induced by hydroxyurea in human diploid fibroblasts

Hydroxyurea was found to inhibit the growth of human diploid fibroblasts, which resulted in senescence-like changes both in morphology and replicative potential similar to the replicative senescence. SA-beta-gal activity, a typical characteristic of the replicative senescence was also induced through a long-term treatment of the presenescent cells with 400-800 microgM of hydroxyurea for about 3 weeks. In addition, we determined the levels of cyclin-dependent kinase inhibitors, p21(Waf1) and p16(INK4a), and the p53 tumor suppressor in order to monitor its effect on cell cycle and stress responses. We observed a great induction of both p53 and p21(Waf1), but not of p16(INK4a) in the premature senescent cells. UV-irradiation of the premature senescent cells showed a decreased level of DNA fragmentation presumably ascribed to the reduced activation of stress-activated protein kinases. These results suggest that a chronic hydroxyurea treatment induces the cellular senescence in association with the induction of p53 and p21(Waf1).     

Aberrant expression of G1-phase cell cycle regulators in flat and exophytic adenomas of the human colon

BACKGROUND & AIMS: The G1/S-phase controlling mechanism known as the RB pathway is commonly deregulated in human malignancies. Here, the abundance and localization of key components of the retinoblastoma (RB) pathway were determined in exophytic and flat colorectal adenomas. METHODS: Samples of normal colonic mucosa (n = 41) and flat (n = 45) and exophytic (n = 26) adenomas were examined immunohistochemically using antibodies to cyclins D1, D2, D3, cyclin-dependent kinase (CDK) 4, retinoblastoma protein (pRB), and the CDK inhibitors p16INK4a, p18INK4c, and p19INK4d. RESULTS: In normal colonic epithelium, cyclin D2 was undetectable; expression of cyclin D1, CDK4, and pRB correlated with proliferation; and p16, p18, p19, and cyclin D3 were most abundant in quiescent, differentiated cells. Adenomas showed elevated expression of cyclin D1 and pRB, frequent induction of cyclin D2, and absence of p16. No obvious abnormalities were found for p18, p19, or cyclin D3. Overexpressed cyclin D2 was more common among exophytic and pRB among flat adenomas, respectively. Elevated cyclin D1, D2, and CDK4 correlated with enhanced dysplasia. CONCLUSIONS: Aberrant expression of cyclins D1, D2, CDK4, p16, and pRB occur in significant subsets of exophytic and flat adenomas, particularly among cases with high-grade dysplasia. Such defects of the RB pathway may perturb cell-cycle control and thereby contribute an early step in colorectal tumorigenesis.     

Activation of p38 MAP kinase and stress signalling in fibroblasts from the progeroid Rothmund–Thomson syndrome

Rothmund–Thomson fibroblasts had replicative lifespans and growth rates within the range for normal fibroblasts; however, they show elevated levels of the stress-associated p38 MAP kinase, suggestive of stress during growth. Treatment with the p38 MAP kinase inhibitor SB203580 increased both lifespan and growth rate, as did reduction of oxidative stress using low oxygen in some strains. At replicative senescence p53, p21^WAF1 and p16^INK4A levels were elevated, and abrogation of p53 using shRNA knockdown allowed the cells to bypass senescence. Ectopic expression of human telomerase allowed Rothmund–Thomson fibroblasts to bypass senescence. However, activated p38 was still present, and continuous growth for some telomerised clones required either a reduction in oxidative stress or SB203580 treatment. Overall, the evidence suggests that replicative senescence in Rothmund–Thomson cells resembles normal senescence in that it is telomere driven and p53 dependent. However, the lack of RECQL4 leads to enhanced levels of stress during cell growth that may lead to moderate levels of stress-induced premature senescence. As replicative senescence is believed to underlie human ageing, a moderate level of stress-induced premature senescence and p38 activity may play a role in the relatively mild ageing phenotype seen in Rothmund–Thomson.

Electronic supplementary material

The online version of this article (doi:10.1007/s11357-012-9476-9) contains supplementary material, which is available to authorized users.     

Relation between maximum replicative capacity and oxidative stress-induced responses in human skin fibroblasts in vitro

Cellular senescence, an important factor in ageing phenotypes, can be induced by replicative exhaustion or by stress. We investigated the relation between maximum replicative capacity, telomere length, stress-induced cellular senescence, and apoptosis/cell death in human primary fibroblast strains obtained from nonagenarians of the Leiden 85-plus Study. Fibroblast strains were cultured until replicative senescence and stressed with rotenone at low passage. Telomere length, senescence-associated-β-galactosidase activity, sub-G1 content, and Annexin-V/PI positivity were measured in nonstressed and stressed conditions. Fibroblast strains with a higher replicative capacity had longer telomeres (p =?.054). In nonstressed conditions, replicative capacity was not associated with β-gal activity (p =?.07) and negatively with sub-G1 (p =?.008). In rotenone-stressed conditions, replicative capacity was negatively associated with β-gal activity (p =?.034) and positively with sub-G1 (p =?.07). Summarizing, fibroblast strains with a higher maximum replicative capacity have longer telomeres, are less prone to go into stress-induced cellular senescence, and more prone to die after stress.     

Absence of R24C Mutation of the <Emphasis Type="Italic">CDK4</Emphasis> Gene in Leukemias and Solid Tumors

A mutation of the p16(INK4a)-binding domain of the cyclin dependent kinase 4 (CDK4) gene, R24C, has been reported in some cases of melanoma. This mutation prevented binding of the CDK4 inhibitor p16(INK48) to CDK4. To determine the relevance of the mutation, we performed polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis in diverse types of human leukemias and solid tumors. No mobility shifts indicating sequence alterations were observed in 273 tumors and 49 cell lines from diverse kinds of tumors These results suggest that in contrast to melanoma, in many other types of human neoplasms the mutation of the CDK4 gene is very rare. To better understand these findings, we randomly mutagenized the CDK4 gene and used the yeast two-hybrid method to screen for CDK4 mutants that had lost the ability to bind to p16(INK4a). Sequence analysis and in vitro kinase assays showed that most of the mutations that disrupted interactions with p16(INK4) also knocked out the activity of CDK4. This result may explain the rareness of CDK4 mutations in human tumors.     

Homeostatic cell-cycle control by BLyS: Induction of cell-cycle entry but not G1/S transition in opposition to p18INK4c and p27Kip1

Cell-cycle entry is critical for homeostatic control in physiologic response of higher organisms but is not well understood. The antibody response begins with induction of naive mature B cells, which are naturally arrested in G(0)/G(1) phase of the cell cycle, to enter the cell cycle in response to antigen and cytokine. BLyS (BAFF), a cytokine essential for mature B cell development and survival, is thought to act mainly by attenuation of apoptosis. Here, we show that BLyS alone induces cell-cycle entry and early G(1) cell-cycle progression, but not S-phase entry, in opposition to the cyclin-dependent kinase inhibitors p18(INK4c). Independent of its survival function, BLyS enhances the synthesis of cyclin D2, in part through activation of NF-kappaB, as well as CDK4 and retinoblastoma protein phosphorylation. By convergent activation of the same cell-cycle regulators in opposition to p18(INK4c), B cell receptor signaling induces cell-cycle entry and G(1) progression in synergy with BLyS, but also DNA replication. The failure of BLyS to induce S-phase cell-cycle entry lies in its inability to increase cyclin E and reduce p27(Kip1) expression. Antagonistic cell-cycle regulation by BLyS and p18(INK4c) is functionally linked to apoptotic control and conserved from B cell activation in vitro to antibody response in vivo, further indicating a physiologic role in homeostasis.     

Functional regulation of D-type cyclins by insulin-like growth factor-I and serum in multiple myeloma cells

D-type cyclin genes are universally dysregulated in multiple myeloma (MM), but the functional consequences are unclear as D-type cyclin gene expression does not correlate with proliferation or disease progression. We examined the protein expression and regulation of D-type cyclins and other cell cycle regulators in human myeloma cell lines and primary CD138(+) plasma cells (PCs). Cyclin D1, cyclin D2, cyclin dependent kinase (CDK) 4, CDK6, p27(Kip1) p18(INK4C) and retinoblastoma protein (pRb) were absent in normal PCs, heterogeneously expressed in primary MM cells and positively correlated with disease activity/progression. Cyclins D1 and D2 complexed with both CDK4 and CDK6, suggesting that both phosphorylate pRb in MM. Furthermore, cyclin D2 expressed via either t(14;16) or t(4;14) IgH translocations was functionally upregulated by fetal calf serum or insulin-like growth factor-I, leading to pRb phosphorylation and cell cycle entry/progression, and in some cases inversely correlated with p27(Kip1). However, pRb phosphorylation and cell cycle progression mediated by cyclin D1 expressed via t(11;14) was less dependent on exogenous stimuli. These data suggest that the presence or absence of specific IgH translocations underlying aberrant D-type cyclin expression may influence their response to mitogens in the bone marrow microenvironment. We showed for the first time that D-type cyclins are functionally regulated in MM, differentially responsive to exogenous growth factors and upregulated with disease progression.