Biomedical subjects

Xiaohu Tang

Publications and source records attributed to Xiaohu Tang.

9 recordsLinked to original sources

PubMed2006

Mutant p53 protects cells from 12-O-tetradecanoylphorbol-13-acetate-induced death by attenuating activating transcription factor 3 induction.

Mutations in p53 are ubiquitous in human tumors. Some p53 mutations not only result in loss of wild-type (WT) activity but also grant additional functions, termed "gain of function." In this study, we explore how the status of p53 affects the immediate response gene activating transcription factor 3 (ATF3) in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-protein kinase C (PKC) pathway. We show that high doses of TPA induce ATF3 in a WT p53-independent manner correlating with PKCs depletion and cell death. We show that cells harboring mutant p53 have attenuated ATF3 induction and are less sensitive to TPA-induced death compared with their p53-null counterparts. Mutagenesis analysis of the ATF3 promoter identified the regulatory motifs cyclic AMP-responsive element binding protein/ATF and MEF2 as being responsible for the TPA-induced activation of ATF3. Moreover, we show that mutant p53 attenuates ATF3 expression by two complementary mechanisms. It interacts with the ATF3 promoter and influences its activity via the MEF2 site, and additionally, it attenuates transcriptional expression of the ATF3 activator MEF2D. These data provide important insights into the molecular mechanisms that underlie mutant p53 gain of function.

Activating Transcription Factor 3↗

PubMed2005

Transcriptional programs following genetic alterations in p53, INK4A, and H-Ras genes along defined stages of malignant transformation.

The difficulty to dissect a complex phenotype of established malignant cells to several critical transcriptional programs greatly impedes our understanding of the malignant transformation. The genetic elements required to transform some primary human cells to a tumorigenic state were described in several recent studies. We took the advantage of the global genomic profiling approach and tried to go one step further in the dissection of the transformation network. We sought to identify the genetic signatures and key target genes, which underlie the genetic alterations in p53, Ras, INK4A locus, and telomerase, introduced in a stepwise manner into primary human fibroblasts. Here, we show that these are the minimally required genetic alterations for sarcomagenesis in vivo. A genome-wide expression profiling identified distinct genetic signatures corresponding to the genetic alterations listed above. Most importantly, unique transformation hallmarks, such as differentiation block, aberrant mitotic progression, increased angiogenesis, and invasiveness, were identified and coupled with genetic signatures assigned for the genetic alterations in the p53, INK4A locus, and H-Ras, respectively. Furthermore, a transcriptional program that defines the cellular response to p53 inactivation was an excellent predictor of metastasis development and bad prognosis in breast cancer patients. Deciphering these transformation fingerprints, which are affected by the most common oncogenic mutations, provides considerable insight into regulatory circuits controlling malignant transformation and will hopefully open new avenues for rational therapeutic decisions.

Cell Differentiation↗

PubMed2004

p53-dependent down-regulation of telomerase is mediated by p21waf1.

Inactivation of p53 and activation of telomerase occur in the majority of human cancers, raising the possibility of a link between these two pathways. Overexpression of wild-type p53 down-regulates the enzymatic activity of telomerase in various cancer cell lines through transcriptional repression of its catalytic subunit, human telomerase reverse transcriptase (hTERT). In this study, we re-evaluated the role of p53 in telomerase regulation using isogenic cell lines expressing physiological levels of p53. We demonstrate that endogenous wild-type p53 was able to down-regulate telomerase activity, hTERT mRNA levels, and promoter activity; however, the ability to repress hTERT expression was found to be cell type-specific. The integrity of the DNA-binding core domain, the N-terminal transactivation domain, and the C-terminal oligomerization domains of p53 was essential for hTERT promoter repression, whereas the proline-rich domain and the extreme C terminus were not required. Southwestern and chromatin immunoprecipitation experiments demonstrated lack of p53 binding to the hTERT promoter, raising the possibility of an indirect repressive mechanism. The down-regulation of hTERT promoter activity was abolished by a dominant-negative E2F1 mutant. Mutational analysis identified a specific E2F site responsible for p53-mediated repression. Knockdown of the key p53 transcriptional target, p21, was sufficient to eliminate the p53-dependent repression of hTERT. Inactivation of the Rb family using either viral oncoproteins or RNA interference attenuated the repression. Inhibition of histone deacetylases also interfered with the repression of hTERT by p53. Therefore, our results suggest that repression of hTERT by endogenous p53 is mediated by p21 and E2F.

Base Sequence↗

PubMed2004

Activated p53 suppresses the histone methyltransferase EZH2 gene.

Replicative senescence is an irreversible cell cycle arrest that limits the proliferation of damaged cells and may be an important tumor suppression mechanism in vivo. This process is regulated at critical steps by the tumor suppressor p53. To identify genes that may regulate the senescence process, we performed cDNA microarray analysis of gene expression in senescent, young proliferating, and hTERT-immortalized primary human fibroblasts. The histone methyltransferase (HMTase), EZH2, was specifically downregulated in senescent cells. Activated p53 suppressed EZH2 gene expression through repression of the EZH2 gene promoter. This activity of p53 requires intact p53 transactivation and DNA binding domains. Furthermore, the repression of EZH2 promoter by p53 is dependent on p53 transcriptional target p21(Waf1) inactivating RB/E2F pathways. In addition, the knockdown of EZH2 expression retards cell proliferation and induces G2/M arrest. We suggest that the p53-dependent suppression of EZH2 expression is a novel pathway that contributes to p53-mediated G2/M arrest. EZH2 associated complex possesses HMTase activity and is involved in epigenetic regulation. Activated p53 suppresses EZH2 expression, suggesting a further role for p53 in epigenetic regulation and in the maintenance of genetic stability. Suppression of EZH2 expression in tumors by p53 may lead to novel approaches to control cancer progression.

Base Sequence↗

PubMed2003

Prolonged culture of telomerase-immortalized human fibroblasts leads to a premalignant phenotype.

Telomere shortening in primary human fibroblasts results in replicative senescence, which can be overcome by telomerase (hTERT) overexpression. However, because immortalization is one of the hallmarks of malignant transformation, careful analysis of hTERT-immortalized cells is of crucial importance for understanding both processes. To this end, we infected WI-38 fibroblasts with a retrovirus carrying the hTERT cDNA and analyzed their proliferative behavior during 600 days [ approximately 500 population doublings (PDLs)] of continuous culture. Growth of three independent mass cultures was uniform for approximately 150 PDLs after telomerase infection, followed by a progressive acceleration of growth in two of three cultures. Expression of p16(INK4A) was significantly elevated in the immortalized cells but gradually disappeared during the accelerated growth phase. This alteration correlated with loss of the contact inhibition response and conferred the cells with sensitivity to H-Ras-induced transformation. In contrast, the p53- and pRb-mediated checkpoints such as the DNA damage response, chromosomal stability and entry into quiescence remained intact, irrespective of INK4A locus expression. Importantly, detailed examination of one of the WI-38/hTERT cultures during the accelerated growth phase revealed overexpression of the c-myc and Bmi-1 oncogenes, as well as loss of p14(ARF) expression. Collectively, our results indicate that although hTERT-immortalized cells behave similarly to primary cells during the first 150 PDLs, long-term growth in culture may favor the appearance of clones carrying potentially malignant alterations.

Cell Division↗

PubMed2002

The onset of p53-dependent DNA repair or apoptosis is determined by the level of accumulated damaged DNA.

The p53 tumor suppressor gene plays an important role in both apoptosis and DNA repair pathways that are pivotal for genomic stability. Here we show that the treatment of cells with low doses of gamma-irradiation or cisplatin resulted in an immediate enhancement of p53-dependent DNA repair, measured by base excision repair (BER) activity. However, treatment of cells with high doses of DNA damaging agents resulted in a reduction in p53-dependent DNA repair and in the induction of p53-dependent apoptosis. Analysis of p53 upstream molecular events suggested that regulation of p53-associated DNA repair is ATM-dependent. Furthermore, we observed that while dephosphorylation of Ser376 at the C-terminus of the p53 protein was associated with enhancement in DNA repair, phosphorylation at the N-terminal Ser15 resulted in the reduction in DNA repair. The latter is also in correlation with an enhancement in the specific DNA binding activity and in the induction of apoptosis. Treatment of cells with a caspase inhibitor, prior to the damaging agent-blocked apoptosis, had no effect on the DNA repair pattern. Taken together, this suggests that the decision of cells to induce a p53-dependent DNA repair or apoptosis is most probably controlled by the level of genotoxic agent introduced to cells.

Animals↗

PubMedDate not supplied

Quantitative measurements of charging in a gaseous environment.

Charge accumulation in insulating or semiconducting samples due to electron beam irradiation is one of the key problems in electron microscopy. One of the most promising techniques for reducing the severity of such charging is to surround the sample with a low-pressure atmosphere of a gas. The charging behavior of a number of materials, surrounded by a variety of gases, has been determined to identify the important factors which control charging under these conditions. The magnitude of the surface potential was deduced from an analysis of x-ray spectra from the surface. The relationship between surface charge, gas pressure, and gas type are measured, and the charging reduction efficiency (CRE) is compared.

Journal Article↗

PubMedDate not supplied

A novel technique for visualizing electron beam induced charging.

Charging is one of the most important problems encountered in scanning electron microscopy and as a result this phenomenon it has received a lot of both theoretical and experimental attention. Despite this, many questions remain about the nature and behavior of charging because of the limitations of the experimental techniques available to study it. For example, although it is now straightforward to determine in situ the surface potential of a sample that is charging during irradiation, it is difficult to measure the lateral extent of the charging, or its persistence once the incident beam is switched off. We describe here a simple technique which provides a rapid way of visualizing the temporal and spatial behavior of charging phenomena.

Journal Article↗

PubMedDate not supplied

An experimental model of beam broadening in the variable pressure scanning electron microscope.

In the variable pressure scanning electron microscope (VP-SEM) the incident electrons pass through a gaseous environment and the beam is scattered by these interactions. We show here that the experimental intensity profile of the scattered beam can be described as Gaussian in form to a high level of accuracy. This provides a rapid means of accounting for the effects of beam scatter in imaging and microanalysis because the standard deviation of the Gaussian is a simple function of parameters such as working distance, beam energy, gas type and pressure.

Journal Article↗