Crosstalk of moderate ROS and PARP-1 contributes to sustainable proliferation of conditionally reprogrammed keratinocytes.

Conditionally reprogrammed cell (CRC) technique is a promising model for biomedical and toxicological research. In the present study, our data first demonstrated an increased level of PARP-1 in conditionally reprogrammed human foreskin keratinocytes (CR-HFKs). We then found that PARP inhibitor ABT-888 (ABT), reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC), or combination (ABT + NAC) were able to inhibit cell proliferation, ROS, PARP-1, and ROS related protein, NRF2, and NOX1. Interestingly, knockdown of endogenous PARP-1 significantly inhibited cell proliferation, indicating that the increased PARP-1 expression was critical for CR. Importantly, we found that a moderate level of ROS contributed the cell proliferation and increased PARP-1 since knockdown of PARP-1 also inhibited the ROS. The similar inhibition of cell proliferation, ROS, and expression of PARP-1 and NRF2 proteins was observed when CR-HFKs were treated with hydroquinone (HQ), a key component from skin-lightening products. Moreover, the treatment of HQ plus treatment of ABT, NAC, or combination can further inhibit cell proliferation, ROS, expression of PARP-1, and NRF2 proteins. PARP-1 knockdown inhibited the population doubling (PDL) and treatment of HQ inhibited the PDL further, as well as the change of ROS. Finally, we discovered that pathways including cyclin D1, NRF2, Rb and pRb, CHK2, and p53, were involved in cell proliferation inhibition with HQ. Taken together, our findings demonstrated that crosstalk between ROS and PARP-1 involves in the cell proliferation in CR-HFKs, and that inhibition of CR-HFK proliferation with HQ is through modulating G1 cell cycle arrest.

Conditional cell reprogramming for modeling host-virus interactions and human viral diseases.

Conventional cancer and transformed cell lines are widely used in cancer biology and other fields within biology. These cells usually have abnormalities from the original tumor itself, but may also develop abnormalities due to genetic manipulation, or genetic and epigenetic changes during long-term passages. Primary cultures may maintain lineage functions as the original tissue types, yet they have a very limited life span or population doubling time because of the nature of cellular senescence. Primary cultures usually have very low yields, and the high variability from any original tissue specimens, largely limiting their applications in research. Animal models are often used for studies of virus infections, disease modeling, development of antiviral drugs, and vaccines. Human viruses often need a series of passages in vivo to adapt to the host environment because of variable receptors on the cell surface and may have intracellular restrictions from the cell types or host species. Here, we describe a long-term cell culture system, conditionally reprogrammed cells (CRCs), and its applications in modeling human viral diseases and drug discovery. Using feeder layer coculture in presence of Y-27632 (conditional reprogramming, CR), CRCs can be obtained and rapidly propagated from surgical specimens, core or needle biopsies, and other minimally invasive or noninvasive specimens, for example, nasal cavity brushing. CRCs preserve their lineage functions and provide biologically relevant and physiological conditions, which are suitable for studies of viral entry and replication, innate immune responses of host cells, and discovery of antiviral drugs. In this review, we summarize the applications of CR technology in modeling host-virus interactions and human viral diseases including severe acute respiratory syndrome coronavirus-2 and coronavirus disease-2019, and antiviral discovery.

Fidelity of a PDX-CR model for bladder cancer.

Patient-derived xenografts (PDXs) are widely recognised as a more physiologically relevant preclinical model than standard cell lines, but are expensive and low throughput, have low engraftment rate and take a long time to develop. Our newly developed conditional reprogramming (CR) technology addresses many PDX drawbacks, but lacks many in vivo factors. Here we determined whether PDXs and CRCs of the same cancer origin maintain the biological fidelity and complement each for translational research and drug development. Four CRC lines were generated from bladder cancer PDXs. Short tandem repeat (STR) analyses revealed that CRCs and their corresponding parental PDXs shared the same STRs, suggesting common cancer origins. CRCs and their corresponding parental PDXs contained the same genetic alterations. Importantly, CRCs retained the same drug sensitivity with the corresponding downstream signalling activity as their corresponding parental PDXs. This suggests that CRCs and PDXs can complement each other, and that CRCs can be used for in vitro fast, high throughput and low cost screening while PDXs can be used for in vivo validation and study of the in vivo factors during translational research and drug development.

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.

Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells.

The roles of protection of telomeres 1 (POT1) in human ovarian cancer have not been fully elucidated. Here, we investigated the impact of POT1 knockdown (POT1-KD) on in vitro cell proliferation, tumorigenesis, and histone deacetylase inhibitor (HDACi) response in human ovarian cancer-derived SK-OV3 cells. The POT1 gene was knocked down by infection with POT1 lenti-shRNA. POT1, c-Myc, and hTERT mRNA levels and relative telomere length were determined by qRT-PCR; POT1 protein levels were determined by western blot. The relative telomerase activity levels were detected using qTRAP; cell proliferation was assessed using cumulative population doubling (cPD) experiments. Cell tumorigenicity was evaluated by anchorage-independent cell growth assays, and cell response to HDACi was determined by luminescence cell viability assays. Results indicate that lenti-shRNA-mediated POT1-KD significantly reduced POT1 mRNA and protein expression. POT1-KD immediately downregulated c-Myc expression, which led to the inhibition of cell proliferation, tumorigenesis, and HDACi response. However, after brief suppression, c-Myc expression increased in the medium term, which resulted in enhanced cell proliferation, tumorigenesis, and HDACi response in the POT1-KD cells. Furthermore, we discovered that c-Myc regulated cell proliferation and tumorigenesis via hTERT/telomerase/telomere pathway.

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.

Autophagic degradation of the inhibitory p53 isoform Δ133p53α as a regulatory mechanism for p53-mediated senescence.

Δ133p53α, a p53 isoform that can inhibit full-length p53, is downregulated at replicative senescence in a manner independent of mRNA regulation and proteasome-mediated degradation. Here we demonstrate that, unlike full-length p53, Δ133p53α is degraded by autophagy during replicative senescence. Pharmacological inhibition of autophagy restores Δ133p53α expression levels in replicatively senescent fibroblasts, without affecting full-length p53. The siRNA-mediated knockdown of pro-autophagic proteins (ATG5, ATG7 and Beclin-1) also restores Δ133p53α expression. The chaperone-associated E3 ubiquitin ligase STUB1, which is known to regulate autophagy, interacts with Δ133p53α and is downregulated at replicative senescence. The siRNA knockdown of STUB1 in proliferating, early-passage fibroblasts induces the autophagic degradation of Δ133p53α and thereby induces senescence. Upon replicative senescence or STUB1 knockdown, Δ133p53α is recruited to autophagosomes, consistent with its autophagic degradation. This study reveals that STUB1 is an endogenous regulator of Δ133p53α degradation and senescence, and identifies a p53 isoform-specific protein turnover mechanism that orchestrates p53-mediated senescence.

p53 isoforms regulate aging- and tumor-associated replicative senescence in T lymphocytes.

Cellular senescence contributes to aging and decline in tissue function. p53 isoform switching regulates replicative senescence in cultured fibroblasts and is associated with tumor progression. Here, we found that the endogenous p53 isoforms Δ133p53 and p53ß are physiological regulators of proliferation and senescence in human T lymphocytes in vivo. Peripheral blood CD8+ T lymphocytes collected from healthy donors displayed an age-dependent accumulation of senescent cells (CD28-CD57+) with decreased Δ133p53 and increased p53ß expression. Human lung tumor-associated CD8+ T lymphocytes also harbored senescent cells. Cultured CD8+ blood T lymphocytes underwent replicative senescence that was associated with loss of CD28 and Δ133p53 protein. In poorly proliferative, Δ133p53-low CD8+CD28- cells, reconstituted expression of either Δ133p53 or CD28 upregulated endogenous expression of each other, which restored cell proliferation, extended replicative lifespan and rescued senescence phenotypes. Conversely, Δ133p53 knockdown or p53ß overexpression in CD8+CD28+ cells inhibited cell proliferation and induced senescence. This study establishes a role for Δ133p53 and p53ß in regulation of cellular proliferation and senescence in vivo. Furthermore, Δ133p53-induced restoration of cellular replicative potential may lead to a new therapeutic paradigm for treating immunosenescence disorders, including those associated with aging, cancer, autoimmune diseases, and HIV infection.

Positive feedback between p53 and TRF2 during telomere-damage signalling and cellular senescence.

The telomere-capping complex shelterin protects functional telomeres and prevents the initiation of unwanted DNA-damage-response pathways. At the end of cellular replicative lifespan, uncapped telomeres lose this protective mechanism and DNA-damage signalling pathways are triggered that activate p53 and thereby induce replicative senescence. Here, we identify a signalling pathway involving p53, Siah1 (a p53-inducible E3 ubiquitin ligase) and TRF2 (telomere repeat binding factor 2; a component of the shelterin complex). Endogenous Siah1 and TRF2 were upregulated and downregulated, respectively, during replicative senescence with activated p53. Experimental manipulation of p53 expression demonstrated that p53 induces Siah1 and represses TRF2 protein levels. The p53-dependent ubiquitylation and proteasomal degradation of TRF2 are attributed to the E3 ligase activity of Siah1. Knockdown of Siah1 stabilized TRF2 and delayed the onset of cellular replicative senescence, suggesting a role for Siah1 and TRF2 in p53-regulated senescence. This study reveals that p53, a downstream effector of telomere-initiated damage signalling, also functions upstream of the shelterin complex.

p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence.

The finite proliferative potential of normal human cells leads to replicative cellular senescence, which is a critical barrier to tumour progression in vivo. We show that the human p53 isoforms Delta133p53 and p53beta function in an endogenous regulatory mechanism for p53-mediated replicative senescence. Induced p53beta and diminished Delta133p53 were associated with replicative senescence, but not oncogene-induced senescence, in normal human fibroblasts. The replicatively senescent fibroblasts also expressed increased levels of miR-34a, a p53-induced microRNA, the antisense inhibition of which delayed the onset of replicative senescence. The siRNA (short interfering RNA)-mediated knockdown of endogenous Delta133p53 induced cellular senescence, which was attributed to the regulation of p21(WAF1) and other p53 transcriptional target genes. In overexpression experiments, whereas p53beta cooperated with full-length p53 to accelerate cellular senescence, Delta133p53 repressed miR-34a expression and extended the cellular replicative lifespan, providing a functional connection of this microRNA to the p53 isoform-mediated regulation of senescence. The senescence-associated signature of p53 isoform expression (that is, elevated p53beta and reduced Delta133p53) was observed in vivo in colon adenomas with senescent phenotypes. The increased Delta133p53 and decreased p53beta isoform expression found in colon carcinoma may signal an escape from the senescence barrier during the progression from adenoma to carcinoma.

Immunomodulatory effect of DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane) on complement system and macrophages.

DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane) is responsible for many immuno-dysregulatory functions in exposed animals, but data particularly on complement system and macrophages are limited. In this study we have shown that DDT activates the complement system through the alternative pathway in the absence of any pathogen. A significant (p<0.05) increase in C3b, C3d and C3a generation, and decline in complement hemolytic activity was observed in insecticide exposed sera. The uncontrolled complement consumption reduces the lytic activity of the complement, which enhances the susceptibility to pyogenic infection if the exposure to DDT remains unabated. Further, DDT induced the significant (p<0.05) production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in macrophages and thus contributes inflammatory reactions, cytokine imbalance and immune-dysregulation. These molecular changes in macrophages lead to structural aberrations like heterochromatin condensation, loss of pseudopodia, cytoplasmic vacuolization, DNA fragmentation and hypodiploid nuclei as seen in our study, suggesting apoptosis. However, in presence lipopolysaccharide, DDT induced significant (p<0.05) suppression of TNF-alpha and NO generation, suggestive of impairment of macrophage microbiocidal effects. This study concludes that the functional and structural derangements of macrophages in association with uncontrolled and excessive complement consumption by DDT are perhaps one of the major mechanisms contributing to the immunosuppressive effects of insecticide.

Modulation of CR1 transcript in systemic lupus erythematosus (SLE) by IFN-gamma and immune complex.

Reduced expression of Erythrocyte Complement Receptor 1 (E-CR1) is envisaged to contribute significantly to the pathophysiology of systemic lupus erythematosus (SLE). We determined the levels of CR1 transcript in the neutrophils from 25 untreated patients with active SLE and 25 normal healthy individuals and, studied the effect of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and immune complexes (IC) on the same. The study revealed a marked decline in the levels of neutrophil CR1 (N-CR1) transcript in the patients with SLE, and differential pattern of IFN-gamma and IL-4 expression in the neutrophils from normals and patients. Opsonized immune complexes down regulated CR1 transcript in patients and IFN-gamma up regulated the same both in normals and patients. Immune complexes suppressed this effect of IFN-gamma. IL-4 also suppressed the effect of IFN-gamma but effect confined only to the normals. This is the first real-time RT-PCR data comparing the neutrophil CR1 expression in normals and patients with SLE and its modulation by IFN-gamma, IL-4 and immune complexes. IFN-gamma and immune complexes, respectively, emerged as the positive and negative modulators of neutrophil CR1 transcript in SLE.

Identification and functional characterization of a novel unspliced transcript variant of HIC-1 in human cancer cells exposed to adverse growth conditions.

The wild-type p53 gene has been widely implicated in the regulation of hypermethylated in cancer-1 (HIC-1) transcription, a master growth regulatory gene with multiple promoters and, consequently, multiple alternatively spliced transcripts. We investigated the role of p53 (wild type and mutant, both endogenous and exogenous) in modulating the various HIC-1 transcripts. We discovered a novel unspliced HIC-1 transcript, identified as "f" in leukocytes and in the human cell lines U87MG (wild-type p53), U373MG (mutant p53), MCF-7 (wild-type p53), HeLa (p53 degraded by HPV18-E6 oncoprotein), and Saos-2 (p53 null). This transcript is initiated from a new transcription start site and has an intervening stop codon that would result in a possibly truncated 22-amino-acid polypeptide. When U87MG (wild-type p53) and MCF-7 cells (wild-type p53) were exposed to adverse growth conditions of serum starvation or treated with the chemotherapeutic agent cisplatin, cells underwent apoptosis and cell cycle arrest accompanied by increase in p53 and HIC-1 transcript levels. Although the increase of the HIC-1-spliced transcripts followed the increase of p53, increase in f transcript coincided with declining p53 and HIC-1 transcript and protein levels. Moreover, the levels of HIC-1 f transcript were not induced by exogenously transfected wild-type p53 in p53-mutated U373MG and p53-null Saos-2 cells, unlike the spliced transcripts that code for full-length HIC-1 protein. These findings suggest a working model wherein the status of f transcript, which is not under direct transcriptional control of wild-type p53, may influence the level of HIC-1 protein in cancer cells.