Association of p16INK4a and pRb inactivation with immortalization of human cells

Tsutsui, Takeki

doi:10.1093/carcin/23.12.2111

Cited by 50 publications

(49 citation statements)

References 32 publications

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“…There is also a growing body of evidence indicating that expression of catalytically active telomerase is insufficient for immortalization of some strains of human fibroblasts. Our previous study, as well as independent investigations, demonstrated that several different stains of fibroblasts expressing hTERT ceased proliferating after a significantly extended lifespan (32)(33)(34)(35). In our study of hTERT-transduced MRC5 lung fibroblasts (MRC5hTERT), growth arrest was not permanent, as subsets of mass cultured cells eventually resumed proliferation, and immortal cell lines were established.…”

mentioning

confidence: 52%

Inactivation of p16 , with Retention of pRB and p53/p21 Function, in Human MRC5 Fibroblasts That Overcome a Telomere-independent Crisis during Immortalization

Taylor

James

Schuller

et al. 2004

Journal of Biological Chemistry

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Recent investigations, including our own, have shown that specific strains of fibroblasts expressing telomerase reverse transcriptase (hTERT) have an extended lifespan, but are not immortal. We previously demonstrated that hTERT-transduced MRC5 fetal lung fibroblasts (MRC5hTERTs) bypassed senescence but eventually succumbed to a second mortality barrier (crisis). In the present study, 67 MRC5hTERT clones were established by limiting dilution of a mass culture. Whereas 39/67 clones had an extended lifespan, all 39 extended lifespan clones underwent crisis. 11 of 39 clones escaped crisis and were immortalized. There was no apparent relationship between the fate of clones at crisis and the level of telomerase activity. Telomeres were hyperextended in the majority of the clones analyzed. There was no difference in telomere length of pre-crisis compared with post-crisis and immortal clones, indicating that hyperextended telomeres were conducive for immortalization and confirming that crisis was independent of telomere length. Immortalization of MRC5hTERT cells was associated with repression of the cyclin-dependent kinase inhibitor p16 INK4a and up-regulation of pRB. However, the regulation of pRB phosphorylation and the response of the p53/p21 cip1/waf1 pathway were normal in immortal cells subject to genotoxic stress. Overexpression of oncogenic ras failed to de-repress p16 INK4a in immortal cells. Furthermore, expression of ras enforced senescent-like growth arrest in p16 INK4a -positive, but not p16 INK4a -negative MRC5hTERT cells. Immortal cells expressing ras formed small, infrequent colonies in soft agarose, but were non-tumorigenic. Overall, these results implicate the inactivation of p16 INK4a as a critical event for overcoming telomere-independent crisis, immortalizing MRC5 fibroblasts and overcoming ras-induced premature senescence.

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mentioning

confidence: 52%

Inactivation of p16 , with Retention of pRB and p53/p21 Function, in Human MRC5 Fibroblasts That Overcome a Telomere-independent Crisis during Immortalization

Taylor

James

Schuller

et al. 2004

Journal of Biological Chemistry

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“…Some authors have noted that hTERT þ cells express a normal cell-cycle arrest and checkpoint protein response to specific challenges including DNA damaging agents (including chromium) (Jiang et al, 1999;Morales et al, 1999;Pritchard et al, 2001;Wood et al, 2001;Gorbunova et al, 2002). In contrast, other authors have reported that there is a loss of p16INK4a and hyperphosphorylation of pRb in hTERT þ cells (Tsutsui et al, 2002;Piboonniyom et al, 2003) and in one cell line a mutation in p53 and abnormal G1 checkpoint (Noble et al, 2004).…”

Section: Metal-induced Genomic Instabilitymentioning

confidence: 99%

Effects of hTERT on metal ion-induced genomic instability

et al. 2006

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There is currently a great interest in delayed chromosomal and other damaging effects of low-dose exposure to a variety of pollutants which appear collectively to act through induction of stress-response pathways related to oxidative stress and ageing. These have been studied mostly in the radiation field but evidence is accumulating that the mechanisms can also be triggered by chemicals, especially heavy metals. Humans are exposed to metals, including chromium (Cr) (VI) and vanadium (V) (V), from the environment, industry and surgical implants. Thus, the impact of low-dose stress responses may be larger than expected from individual toxicity projections. In this study, a short (24 h) exposure of human fibroblasts to low doses of Cr (VI) and V (V) caused both acute chromosome damage and genomic instability in the progeny of exposed cells for at least 30 days after exposure. Acutely, Cr (VI) caused chromatid breaks without aneuploidy while V (V) caused aneuploidy without chromatid breaks. The longerterm genomic instability was similar but depended on hTERT positivity. In telomerase-negative hTERTÀ cells, Cr (VI) and V (V) caused a long lasting and transmissible induction of dicentric chromosomes, nucleoplasmic bridges, micronuclei and aneuploidy. There was also a long term and transmissible reduction of clonogenic survival, with an increased b-galactosidase staining and apoptosis. This instability was not present in telomerasepositive hTERT þ cells. In contrast, in hTERT þ cells the metals caused a persistent induction of tetraploidy, which was not noted in hTERTÀ cells. The growth and survival of both metal-exposed hTERT þ and hTERTÀ cells differed if they were cultured at subconfluent levels or plated out as colonies. Genomic instability is considered to be a driving force towards cancer. This study suggests that the type of genomic instability in human cells may depend critically on whether they are telomerase-positive or -negative and that their sensitivities to metals could depend on whether they are clustered or diffuse.

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“…63,64 Conversely, breaking the senescence barrier in order to immortalize cells requires the inactivation of these cell cycle inhibitory genes. 65,66 The latter mechanism likely accounts for the immortalization of AP-1060 cells after ENU mutagenization, since compared to AP-1060S cells, the established line cells showed a marked derepression of genes associated with cell cycle progression and DNA synthesis (Table 3), but with little or no secondary change in telomere length or telomerase activity (Figure 2).…”

Section: Apl Cell Line Ap-1060mentioning

confidence: 99%

Acute promyelocytic leukemia cell line AP-1060 established as a cytokine-dependent culture from a patient clinically resistant to all-trans retinoic acid and arsenic trioxide

et al. 2004

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AP-1060 is a newly established acute promyelocytic leukemia (APL) cell line from a multiple-relapse patient clinically resistant to both all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). The line was initially derived as a granulocyte colonystimulating factor-dependent strain that underwent replicative senescence and, following ethylnitrosourea treatment, as a phenotypically similar immortalized line. Immortalization was associated with broadened cytokine sensitivity but not growth autonomy, in contrast to three previously derived APL lines. Both the AP-1060 strain and line had shortened telomeres and low telomerase activity, while the line had higher expression of many genes associated with macromolecular synthesis. The karyotype was 46,XY,t(3;14)(p21.1;q11.2),t(15;17)(q22;q11) [100%]; the unique t(3;14) was observed in 4/9 t(15;17)-positive metaphase cells at previous relapse on ATRA therapy. The PML-RARa mRNA harbored a missense mutation in the RARaregion ligand-binding domain (Pro900Ser). This was associated with a right-shift and sharpening of the ATRA-induced maturation response compared to ATRA-sensitive NB4 cells, which corresponded to the transcriptional activation by PML-RARaPro900Ser of a cotransfected ATRA-targeted reporter vector in COS-1 cells. AP-1060 also manifested relative resistance to ATO-induced apoptosis at X1 lM, while 0.25 lM ATO stimulated limited atypical maturation. These findings suggest that AP-1060 will be useful for further assessing molecular elements involved in APL progression and drug response/resistance.

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Association of p16INK4a and pRb inactivation with immortalization of human cells

Cited by 50 publications

References 32 publications

Inactivation of p16 , with Retention of pRB and p53/p21 Function, in Human MRC5 Fibroblasts That Overcome a Telomere-independent Crisis during Immortalization

Inactivation of p16 , with Retention of pRB and p53/p21 Function, in Human MRC5 Fibroblasts That Overcome a Telomere-independent Crisis during Immortalization

Effects of hTERT on metal ion-induced genomic instability

Acute promyelocytic leukemia cell line AP-1060 established as a cytokine-dependent culture from a patient clinically resistant to all-trans retinoic acid and arsenic trioxide

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