Enhancing chimeric antigen receptor T cell therapy by modulating the p53 signaling network with Δ133p53α.

Chimeric antigen receptor (CAR) T cell dysfunction is a major barrier to achieving lasting remission in hematologic cancers, especially in chronic lymphocytic leukemia (CLL). We have shown previously that Δ133p53α, an endogenous isoform of the human TP53 gene, decreases in expression with age in human T cells, and that reconstitution of Δ133p53α in poorly functional T cells can rescue proliferation [A. M. Mondal et al., J. Clin. Invest. 123, 5247-5257 (2013)]. Although Δ133p53α lacks a transactivation domain, it can form heterooligomers with full-length p53 and modulate the p53-mediated stress response [I. Horikawa et al., Cell Death Differ. 24, 1017-1028 (2017)]. Here, we show that constitutive expression of Δ133p53α potentiates the anti-tumor activity of CD19-directed CAR T cells and limits dysfunction under conditions of high tumor burden and metabolic stress. We demonstrate that Δ133p53α-expressing CAR T cells exhibit a robust metabolic phenotype, maintaining the ability to execute effector functions and continue proliferating under nutrient-limiting conditions, in part due to upregulation of critical biosynthetic processes and improved mitochondrial function. Importantly, we show that our strategy to constitutively express Δ133p53α improves the anti-tumor efficacy of CAR T cells generated from CLL patients that previously failed CAR T cell therapy. More broadly, our results point to the potential role of the p53-mediated stress response in limiting the prolonged antitumor functions required for complete tumor clearance in patients with high disease burden, suggesting that modulation of the p53 signaling network with Δ133p53α may represent a translationally viable strategy for improving CAR T cell therapy.

EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in hTERT-transduced normal human cells.

BACKGROUND: Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells. METHODS: Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes. RESULTS: The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated ß-galactosidase (SA-ß-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice. CONCLUSIONS: Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.

Targeting cellular senescence in cancer and aging: roles of p53 and its isoforms.

Cellular senescence and the associated secretory phenotype (SASP) promote disease in the aged population. Targeting senescent cells by means of removal, modulation of SASP or through cellular reprogramming represents a novel therapeutic avenue for treating cancer- and age-related diseases such as neurodegeneration, pulmonary fibrosis and renal disease. Cellular senescence is partly regulated by the TP53 gene, a critical tumor suppressor gene which encodes 12 or more p53 protein isoforms. This review marks a significant milestone of 40 years of Carcinogenesis publication history and p53 research and 15 years of p53 isoform research. The p53 isoforms are produced through initiation at alternative transcriptional and translational start sites and alternative mRNA splicing. These truncated p53 isoform proteins are endogenously expressed in normal human cells and maintain important functional roles, including modulation of full-length p53-mediated cellular senescence, apoptosis and DNA repair. In this review, we discuss the mechanisms and functions of cellular senescence and SASP in health and disease, the regulation of cellular senescence by p53 isoforms, and the therapeutic potential of targeting cellular senescence to treat cancer- and age-associated diseases.

Cellular Senescence: Mechanisms, Morphology, and Mouse Models.

Cellular senescence is a cell cycle arrest in damaged or aged cells. Although this represents a critical mechanism of tumor suppression, persistence of senescent cells during aging induces chronic inflammation and tissue dysfunction through the adoption of the senescence-associated secretory phenotype (SASP). This has been shown to promote the progression of age-associated diseases such as Alzheimer's disease, pulmonary fibrosis, and atherosclerosis. As the global population ages, the role of cellular senescence in disease is becoming a more critical area of research. In this review, mechanisms, biomarkers, and pathology of cellular senescence and SASP are described with a brief discussion of literature supporting a role for cellular senescence in veterinary diseases. Cell culture and mouse models used in senescence studies are also reviewed including the senescence-accelerated mouse-prone (SAMP), senescence pathway knockout mice (p53, p21 [CDKN1A], and p16 [CDKN2A]), and the more recently developed senolysis mice, which allow for direct visualization and elimination (or lysis) of senescent cells in live mice (p16-3MR and INK-ATTAC). These and other mouse models have demonstrated the importance of cellular senescence in embryogenesis and wound healing but have also identified a therapeutic benefit for targeting persistent senescent cells in age-associated diseases including neurodegeneration, diabetes, and cardiac fibrosis.

Distinct functions of wild-type and R273H mutant Δ133p53α differentially regulate glioblastoma aggressiveness and therapy-induced senescence.

Despite being mutated in 92% of TP53 mutant cancers, how mutations on p53 isoforms affect their activities remain largely unknown. Therefore, exploring the effect of mutations on p53 isoforms activities is a critical, albeit unexplored area in the p53 field. In this article, we report for the first time a mutant Δ133p53α-specific pathway which increases IL4I1 and IDO1 expression and activates AHR, a tumor-promoting mechanism. Accordingly, while WT Δ133p53α reduces apoptosis to promote DNA repair, mutant R273H also reduces apoptosis but fails to maintain genomic stability, increasing the risks of accumulation of mutations and tumor's deriving towards a more aggressive phenotype. Furthermore, using 2D and 3D spheroids culture, we show that WT Δ133p53α reduces cell proliferation, EMT, and invasion, while the mutant Δ133p53α R273H enhances all three processes, confirming its oncogenic potential and strongly suggesting a similar in vivo activity. Importantly, the effects on cell growth and invasion are independent of mutant full-length p53α, indicating that these activities are actively carried by mutant Δ133p53α R273H. Furthermore, both WT and mutant Δ133p53α reduce cellular senescence in a senescence inducer-dependent manner (temozolomide or radiation) because they regulate different senescence-associated target genes. Hence, WT Δ133p53α rescues temozolomide-induced but not radiation-induced senescence, while mutant Δ133p53α R273H rescues radiation-induced but not temozolomide-induced senescence. Lastly, we determined that IL4I1, IDO1, and AHR are significantly higher in GBMs compared to low-grade gliomas. Importantly, high expression of all three genes in LGG and IL4I1 in GBM is significantly associated with poorer patients' survival, confirming the clinical relevance of this pathway in glioblastomas. These data show that, compared to WT Δ133p53α, R273H mutation reorientates its activities toward carcinogenesis and activates the oncogenic IL4I1/IDO1/AHR pathway, a potential prognostic marker and therapeutic target in GBM by combining drugs specifically modulating Δ133p53α expression and IDO1/Il4I1/AHR inhibitors.

Distinct functions of wild-type and R273H mutant Δ133p53α differentially regulate glioblastoma aggressiveness and therapy-induced senescence.

Mutations effects on p53 isoforms' activities remain largely unknown, although they are mutated in 92% of TP53 mutant cancers. Therefore, exploring the effect of mutations on p53 isoforms activities is a critical, albeit unexplored area in the p53 field. In this article, we report for the first time a mutant Δ133p53α-specific pathway which increases IL4I1 and IDO1 expression and activates AHR, a tumor-promoting mechanism. Accordingly, mutant Δ133p53α R273H increases glioblastoma cancer cells proliferation and invasion while the WT does not. Furthermore, while WT Δ133p53α reduces apoptosis to promote DNA repair, the mutant also reduces apoptosis but fails to maintain genomic stability.Furthermore, both WT and mutant Δ133p53α reduce cellular senescence in a senescence inducer-dependent manner (temozolomide or radiation) because they regulate different senescence-associated target genes. Hence, WT Δ133p53α rescues temozolomide-induced but not radiation-induced senescence, while mutant Δ133p53α R273H rescues radiation-induced but not temozolomide-induced senescence. Lastly, using TCGA data, we determined that IL4I1, IDO1 and AHR are significantly higher in GBMs compared to LGGs. IL4I1 expression is increased in mutant TP53 LGGs and GBMs, although only significantly in LGG. Importantly, high expression of all three genes in LGG and IL4I1 in GBM is significantly associated with poorer patients' survival. These data show that, compared to WT Δ133p53α, R273H mutation reorientates its activities toward carcinogenesis and activates the oncogenic IL4I1/IDO1/AHR pathway, a potential prognostic marker and therapeutic target in GBM by combining drugs specifically modulating Δ133p53α expression and IDO1/Il4I1/AHR inhibitors.

MiR-21 is an EGFR-regulated anti-apoptotic factor in lung cancer in never-smokers.

Fifteen percent of lung cancer cases occur in never-smokers and show characteristics that are molecularly and clinically distinct from those in smokers. Epidermal growth factor receptor (EGFR) gene mutations, which are correlated with sensitivity to EGFR-tyrosine kinase inhibitors (EGFR-TKIs), are more frequent in never-smoker lung cancers. In this study, microRNA (miRNA) expression profiling of 28 cases of never-smoker lung cancer identified aberrantly expressed miRNAs, which were much fewer than in lung cancers of smokers and included miRNAs previously identified (e.g., up-regulated miR-21) and unidentified (e.g., down-regulated miR-138) in those smoker cases. The changes in expression of some of these miRNAs, including miR-21, were more remarkable in cases with EGFR mutations than in those without these mutations. A significant correlation between phosphorylated-EGFR (p-EGFR) and miR-21 levels in lung carcinoma cell lines and the suppression of miR-21 by an EGFR-TKI, AG1478, suggest that the EGFR signaling is a pathway positively regulating miR-21 expression. In the never-smoker-derived lung adenocarcinoma cell line H3255 with mutant EGFR and high levels of p-EGFR and miR-21, antisense inhibition of miR-21 enhanced AG1478-induced apoptosis. In a never-smoker-derived adenocarcinoma cell line H441 with wild-type EGFR, the antisense miR-21 not only showed the additive effect with AG1478 but also induced apoptosis by itself. These results suggest that aberrantly increased expression of miR-21, which is enhanced further by the activated EGFR signaling pathway, plays a significant role in lung carcinogenesis in never-smokers, as well as in smokers, and is a potential therapeutic target in both EGFR-mutant and wild-type cases.

Δ133p53α Protects Human Astrocytes from Amyloid-beta Induced Senescence and Neurotoxicity.

Cellular senescence is an important contributor to aging and age-related diseases such as Alzheimer's disease (AD). Senescent cells are characterized by a durable cell proliferation arrest and the acquisition of a proinflammatory senescence-associated secretory phenotype (SASP), which participates in the progression of neurodegenerative disorders. Clearance of senescent glial cells in an AD mouse model prevented cognitive decline suggesting pharmacological agents targeting cellular senescence might provide novel therapeutic approaches for AD. Δ133p53α, a natural protein isoform of p53, was previously shown to be a negative regulator of cellular senescence in primary human astrocytes, with clinical implications from its diminished expression in brain tissues from AD patients. Here we show that treatment of proliferating human astrocytes in culture with amyloid-beta oligomers (Aß), an endogenous pathogenic agent of AD, results in reduced expression of Δ133p53α, as well as induces the cells to become senescent and express proinflammatory SASP cytokines such as IL-6, IL-1ß and TNFα. Our data suggest that Aß-induced astrocyte cellular senescence is associated with accelerated DNA damage, and upregulation of full-length p53 and its senescence-inducing target gene p21WAF1. We also show that exogenously enhanced expression of Δ133p53α rescues human astrocytes from Aß-induced cellular senescence and SASP through both protection from DNA damage and dominant-negative inhibition of full-length p53, leading to inhibition of Aß-induced, astrocyte-mediated neurotoxicity. The results presented here demonstrate that Δ133p53α manipulation could modulate cellular senescence in the context of AD, possibly opening new therapeutic avenues.

Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks.

Humans and animals undergo ageing, and although their primary cells undergo cellular senescence in culture, the relationship between these two processes is unclear. Here we show that gamma-H2AX foci (gamma-foci), which reveal DNA double-strand breaks (DSBs), accumulate in senescing human cell cultures and in ageing mice. They colocalize with DSB repair factors, but not significantly with telomeres. These cryptogenic gamma-foci remain after repair of radiation-induced gamma-foci, suggesting that they may represent DNA lesions with unrepairable DSBs. Thus, we conclude that accumulation of unrepairable DSBs may have a causal role in mammalian ageing.

Astrocyte senescence and SASP in neurodegeneration: tau joins the loop.

Tau accumulation is a core component of Alzheimer's disease and other neurodegenerative tauopathies. While tau's impact on neurons is a major area of research, the effect of extracellular tau on astrocytes is largely unknown. This article summarizes our recent studies showing that astrocyte senescence plays a critical role in neurodegenerative diseases and integrates extracellular tau into the regulatory loop of senescent astrocyte-mediated neurotoxicity. Human astrocytes in vitro undergoing senescence were shown to acquire the inflammatory senescence-associated secretory phenotype (SASP) and toxicity to neurons, which may recapitulate aging- and disease-associated neurodegeneration. Here, we show that human astrocytes exposed to extracellular tau in vitro also undergo cellular senescence and acquire a neurotoxic SASP (e.g. IL-6 secretion), with oxidative stress response (indicated by upregulated NRF2 target genes) and a possible activation of inflammasome (indicated by upregulated ASC and IL-1ß). These findings suggest that senescent astrocytes induced by various conditions and insults, including tau exposure, may represent a therapeutic target to inhibit or delay the progression of neurodegenerative diseases. We also discuss the pathological activity of extracellular tau in microglia and astrocytes, the disease relevance and diversity of tau forms, therapeutics targeting senescence in neurodegeneration, and the roles of p53 and its isoforms in astrocyte-mediated neurotoxicity and neuroprotection.

Balancing and Differentiating p53 Activities toward Longevity and No Cancer?

Aging and death of cells (cellular senescence and apoptosis, respectively), triggered by or associated with cellular stress and DNA damage, impair organ function and homeostasis, leading to organismal aging and death. On the other hand, defects in physiologic regulations of cellular aging and death (escape from cellular senescence and failed apoptosis of severely damaged cells) contribute to uncontrolled cell division and genetic instability in cancer. In an oversimplified scenario, p53, an inducer of cellular senescence and apoptosis, may thus unfavorably contribute to aging and favorably suppress tumorigenesis. However, physiologic mechanisms should exist and therapeutic approaches may be developed to balance between aging and tumor suppression, for example, by differentially regulating cellular senescence, apoptosis, and other p53-mediated biological processes, such as DNA repair, autophagy, and energy metabolism. Possible mechanisms for such differential regulation of different subsets of p53 target genes may involve posttranslational modifications (e.g., phosphorylation and acetylation) and DNA binding cooperativity of p53. In this issue of Cancer Research, Timofeev and colleagues show that a previously uncharacterized phosphorylation in the p53 core DNA-binding domain regulates the DNA binding cooperativity and transcriptional activity of p53. Their mice deficient for this p53 phosphorylation were resistant to spontaneous and induced tumorigenesis, while they had shortened lifespan, but did not show progeria-like phenotypes. Prompted by this study, research on p53, aging, and cancer will explore balancing and differentiating different p53 activities toward a challenging goal of achieving longevity with no cancer.See related article by Timofeev et al., p. 5231.

Cytokine Storms in Cancer and COVID-19.

During the COVID-19 pandemic, research on "cytokine storms" has been reinvigorated in the field of infectious disease, but it also has particular relevance to cancer research. Interleukin-6 (IL-6) has emerged as a key component of the immune response to SARS-CoV-2, such that the repurposing of anti-IL-6 therapeutics for COVID-19 is now a major line of investigation, with several ongoing clinical trials. We lay a framework for understanding the role of IL-6 in the context of cancer research and COVID-19 and suggest how lessons learned from cancer research may impact SARS-CoV-2 research and vice versa.

The Δ133p53 Isoforms, Tuners of the p53 Pathway.

The TP53 gene is a critical tumor suppressor and key determinant of cell fate which regulates numerous cellular functions including DNA repair, cell cycle arrest, cellular senescence, apoptosis, autophagy and metabolism. In the last 15 years, the p53 pathway has grown in complexity through the discovery that TP53 differentially expresses twelve p53 protein isoforms in human cells with both overlapping and unique biologic activities. Here, we summarize the current knowledge on the Δ133p53 isoforms (Δ133p53α, Δ133p53ß and Δ133p53γ), which are evolutionary derived and found only in human and higher order primates. All three isoforms lack both of the transactivation domains and the beginning of the DNA-binding domain. Despite the absence of these canonical domains, the Δ133p53 isoforms maintain critical functions in cancer, physiological and premature aging, neurodegenerative diseases, immunity and inflammation, and tissue repair. The ability of the Δ133p53 isoforms to modulate the p53 pathway functions underscores the need to include these p53 isoforms in our understanding of how the p53 pathway contributes to multiple physiological and pathological mechanisms. Critically, further characterization of p53 isoforms may identify novel regulatory modes of p53 pathway functions that contribute to disease progression and facilitate the development of new therapeutic strategies.

Radiation-induced astrocyte senescence is rescued by Δ133p53.

BACKGROUND: Cellular senescence and the senescence-associated secretory phenotype (SASP) may contribute to the development of radiation therapy-associated side effects in the lung and blood vessels by promoting chronic inflammation. In the brain, inflammation contributes to the development of neurologic disease, including Alzheimer's disease. In this study, we investigated the roles of cellular senescence and Δ133p53, an inhibitory isoform of p53, in radiation-induced brain injury. METHODS: Senescent cell types in irradiated human brain were identified with immunohistochemical labeling of senescence-associated proteins p16INK4A and heterochromatin protein Hp1γ in 13 patient cases, including 7 irradiated samples. To investigate the impact of radiation on astrocytes specifically, primary human astrocytes were irradiated and examined for expression of Δ133p53 and induction of SASP. Lentiviral expression of ∆133p53 was performed to investigate its role in regulating radiation-induced cellular senescence and astrocyte-mediated neuroinflammation. RESULTS: Astrocytes expressing p16INK4A and Hp1γ were identified in all irradiated tissues, were increased in number in irradiated compared with untreated cancer patient tissues, and had higher labeling intensity in irradiated tissues compared with age-matched controls. Human astrocytes irradiated in vitro also experience induction of cellular senescence, have diminished Δ133p53, and adopt a neurotoxic phenotype as demonstrated by increased senescence-associated beta-galactosidase activity, p16INK4A, and interleukin (IL)-6. In human astrocytes, Δ133p53 inhibits radiation-induced senescence, promotes DNA double-strand break repair, and prevents astrocyte-mediated neuroinflammation and neurotoxicity. CONCLUSIONS: Restoring expression of the endogenous p53 isoform, ∆133p53, protects astrocytes from radiation-induced senescence, promotes DNA repair, and inhibits astrocyte-mediated neuroinflammation.

Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins.

Sir2 is a NAD+-dependent protein deacetylase that extends lifespan in yeast and worms. This study examines seven human proteins homologous to Sir2 (SIRT1 through SIRT7) for cellular localization, expression profiles, protein deacetylation activity, and effects on human cell lifespan. We found that: 1) three nuclear SIRT proteins (SIRT1, SIRT6, and SIRT7) show different subnuclear localizations: SIRT6 and SIRT7 are associated with heterochromatic regions and nucleoli, respectively, where yeast Sir2 functions; 2) SIRT3, SIRT4, and SIRT5 are localized in mitochondria, an organelle that links aging and energy metabolism; 3) cellular p53 is a major in vivo substrate of SIRT1 deacetylase, but not the other six SIRT proteins; 4) SIRT1, but not the other two nuclear SIRT proteins, shows an in vitro deacetylase activity on histone H4 and p53 peptides; and 5) overexpression of any one of the seven SIRT proteins does not extend cellular replicative lifespan in normal human fibroblasts or prostate epithelial cells. This study supports the notion that multiple human SIRT proteins have evolutionarily conserved and nonconserved functions at different cellular locations and reveals that the lifespan of normal human cells, in contrast to that of lower eukaryotes, cannot be manipulated by increased expression of a single SIRT protein.

p53 isoforms regulate premature aging in human cells.

Cellular senescence is a hallmark of normal aging and aging-related syndromes, including the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS), a rare genetic disorder caused by a single mutation in the LMNA gene that results in the constitutive expression of a truncated splicing mutant of lamin A known as progerin. Progerin accumulation leads to increased cellular stresses including unrepaired DNA damage, activation of the p53 signaling pathway and accelerated senescence. We previously established that the p53 isoforms ∆133p53 and p53ß regulate senescence in normal human cells. However, their role in premature aging is unknown. Here we report that p53 isoforms are expressed in primary fibroblasts derived from HGPS patients, are associated with their accelerated senescence and that their manipulation can restore the replication capacity of HGPS fibroblasts. We found that in near-senescent HGPS fibroblasts, which exhibit low levels of ∆133p53 and high levels of p53ß, restoration of Δ133p53 expression was sufficient to extend replicative lifespan and delay senescence, despite progerin levels and abnormal nuclear morphology remaining unchanged. Conversely, Δ133p53 depletion or p53ß overexpression accelerated the onset of senescence in otherwise proliferative HGPS fibroblasts. Our data indicate that Δ133p53 exerts its role by modulating full-length p53 (FLp53) signaling to extend the replicative lifespan and promotes the repair of spontaneous progerin-induced DNA double-strand breaks (DSBs). We showed that Δ133p53 dominant-negative inhibition of FLp53 occurs directly at the p21/CDKN1A and miR-34a promoters, two p53 senescence-associated genes. In addition, Δ133p53 expression increased the expression of DNA repair RAD51, likely through upregulation of E2F1, a transcription factor that activates RAD51, to promote repair of DSBs. In summary, our data indicate that Δ133p53 modulates p53 signaling to repress progerin-induced early onset of senescence in HGPS cells. Therefore, restoration of ∆133p53 expression may be a novel therapeutic strategy to treat aging-associated phenotypes of HGPS in vivo.

Δ133p53α, a natural p53 isoform, contributes to conditional reprogramming and long-term proliferation of primary epithelial cells.

We previously developed the technique of conditional reprogramming (CR), which allows primary epithelial cells from fresh or cryopreserved specimens to be propagated long-term in vitro, while maintaining their genetic stability and differentiation potential. This method requires a combination of irradiated fibroblast feeder cells and a Rho-associated kinase (ROCK) inhibitor. In the present study, we demonstrate increased levels of full-length p53 and its natural isoform, Δ133p53α, in conditionally reprogrammed epithelial cells from primary prostate, foreskin, ectocervical, and mammary tissues. Increased Δ133p53α expression is critical for CR since cell proliferation is rapidly inhibited following siRNA knockdown of endogenous Δ133p53α. Importantly, overexpression of Δ133p53α consistently delays the onset of cellular senescence of primary cells when cultured under non-CR conditions in normal keratinocyte growth medium (KGM). More significantly, the combination of Δ133p53α overexpression and ROCK inhibitor, without feeder cells, enables primary epithelial cells to be propagated long-term in vitro. We also show that Δ133p53α overexpression induces hTERT expression and telomerase activity and that siRNA knockdown of hTERT causes rapid inhibition of cell proliferation, indicating a critical role of hTERT for mediating the effects of Δ133p53α. Altogether, these data demonstrate a functional and regulatory link between p53 pathways and hTERT expression during the conditional reprogramming of primary epithelial cells.

Downstream E-box-mediated regulation of the human telomerase reverse transcriptase (hTERT) gene transcription: evidence for an endogenous mechanism of transcriptional repression.

Regulation of the hTERT gene encoding the telomerase catalytic subunit plays an important role in human cell senescence, immortalization, and carcinogenesis. By examining the activity of various deleted or mutated hTERT promoter fragments, we show that an E-box element downstream of the transcription initiation site is critical to differential hTERT transcription between the telomerase/hTERT-positive renal cell carcinoma cell line (RCC23) and its telomerase/hTERT-negative counterpart containing a transferred, normal chromosome 3 (RCC23+3). This E-box element mediated repression of hTERT transcription in RCC23+3 but not in RCC23. A copy number-dependent enhancement of the repression suggested active repression, rather than loss of activation, in RCC23+3. Endogenous expression levels of c-Myc or Mad1, which could activate or repress hTERT transcription when overexpressed, did not account for the differential hTERT transcription. Gel mobility shift assays identified the upstream stimulatory factors (USFs) as a major E-box-binding protein complex in both RCC23 and RCC23+3 and, importantly, detected an RCC23+3-specific, E-box-binding factor that was distinct from the USF and Myc/Mad families. The E-box-mediated repression was also active in normal human fibroblasts and epithelial cells and inactive in some, but not all, telomerase/hTERT-positive cancer cells. These findings provide evidence for an endogenous, repressive mechanism that actively functions in telomerase/hTERT-negative normal cells and becomes defective during carcinogenic processes, e.g., by an inactivation of the telomerase repressor gene on chromosome 3.

Δ133p53 represses p53-inducible senescence genes and enhances the generation of human induced pluripotent stem cells.

p53 functions to induce cellular senescence, which is incompatible with self-renewal of pluripotent stem cells such as induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC). However, p53 also has essential roles in these cells through DNA damage repair for maintaining genomic integrity and high sensitivity to apoptosis for eliminating severely damaged cells. We hypothesized that Δ133p53, a physiological inhibitory p53 isoform, is involved in the balanced regulation of self-renewing capacity, DNA damage repair and apoptosis. We examined 12 lines of human iPSC and their original fibroblasts, as well as three ESC lines, for endogenous protein levels of Δ133p53 and full-length p53 (FL-p53), and mRNA levels of various p53 target genes. While FL-p53 levels in iPSC and ESC widely ranged from below to above those in the fibroblasts, all iPSC and ESC lines expressed elevated levels of Δ133p53. The p53-inducible genes that mediate cellular senescence (p21WAF1, miR-34a, PAI-1 and IGFBP7), but not those for apoptosis (BAX and PUMA) and DNA damage repair (p53R2), were downregulated in iPSC and ESC. Consistent with these endogenous expression profiles, overexpression of Δ133p53 in human fibroblasts preferentially repressed the p53-inducible senescence mediators and significantly enhanced their reprogramming to iPSC. The iPSC lines derived from Δ133p53-overexpressing fibroblasts formed well-differentiated, benign teratomas in immunodeficient mice and had fewer numbers of somatic mutations than an iPSC derived from p53-knocked-down fibroblasts, suggesting that Δ133p53 overexpression is non- or less oncogenic and mutagenic than total inhibition of p53 activities. Overexpressed Δ133p53 prevented FL-p53 from binding to the regulatory regions of p21WAF1 and miR-34a promoters, providing a mechanistic basis for its dominant-negative inhibition of a subset of p53 target genes. This study supports the hypothesis that upregulation of Δ133p53 is an endogenous mechanism that facilitates human somatic cells to become self-renewing pluripotent stem cells with maintained apoptotic and DNA repair activities.

Upregulation of the KIAA1199 gene is associated with cellular mortality.

The microcell-mediated transfer of a normal human chromosome 3 induces replicative senescence in otherwise immortal renal cell carcinoma cells. To identify the genes involved in the chromosome 3-induced cellular mortality, we previously performed a cDNA subtraction experiment using the immortal renal cell carcinoma cells (RCC23) and the mortal counterpart with the transferred chromosome 3 (RCC23+3). We here report the cDNA cloning and characterization of one of the differentially expressed genes, which encodes KIAA1199 protein of unknown function. Northern blot and RT-PCR analyses revealed striking upregulation of KIAA1199 mRNA in mortal RCC23+3 compared with immortal RCC23. However, no significant change in KIAA1199 mRNA expression was observed during replicative aging in vitro (from early passage culture to senescent culture) of mortal human cells including RCC23+3, normal fibroblasts and prostate epithelial cells. Interestingly, an immortal fibroblast cell line and two breast cancer cell lines expressed much lower amounts of KIAA1199 mRNA than their normal counterparts. KIAA1199 mRNA is expressed in a wide range of normal human tissues, with the highest level of expression in brain. The gene is located on chromosome band 15q25, where a brain tumor suppressor gene has been mapped. These findings suggest that KIAA1199 gene may play a role in cellular mortality of normal human cells, which counters cell immortalization and carcinogenesis.