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Jennifer A Pietenpol

Publications and source records attributed to Jennifer A Pietenpol.

At least 19 recordsLinked to original sources
PubMed2006

Loss of p63 leads to increased cell migration and up-regulation of genes involved in invasion and metastasis.

p63, a homologue of the tumor suppressor p53, is critical for the development and maintenance of squamous epithelia. p63 is specifically expressed in the basal layers of stratified epithelial tissues and is considered a specific marker for cells of this type. The role of p63 in tumorigenesis remains poorly defined. Numerous studies have highlighted the oncogenic potential of the predominant p63 isoform DeltaNp63alpha; however, data suggest that other p63 proteins can act as tumor suppressors or alter the metastatic potential of tumors. DeltaNp63alpha can act as a transcriptional repressor, but the link between the transcriptional functions of p63 and its biological role is still unclear. In this study, we used a loss-of-function approach to investigate the transcriptional programs controlled by p63. Disruption of p63 in squamous cell lines resulted in down-regulation of transcripts specifically expressed in squamous tissues and a significant alteration of keratinocyte differentiation. Interestingly, we found that disruption of p63 led to up-regulation of markers of nonepithelial tissues (mesenchyme and neural tissue) in both primary and immortalized squamous cells. Many of these up-regulated genes are associated with increased capacity for invasion and metastasis in tumors. Furthermore, loss of p63 expression was accompanied by a shift toward mesenchymal morphology and an increase in motility in primary keratinocytes and squamous cell lines. We conclude that loss of endogenous p63 expression results in up-regulation of genes associated with invasion and metastasis, and predisposes to a loss of epithelial and acquisition of mesenchymal characteristics. These findings have implications for the role of p63 in both development and tumorigenesis.

Carcinoma, Squamous Cell↗
PubMed2006

The DNA binding activity of p53 displays reaction-diffusion kinetics.

The tumor suppressor protein p53 plays a key role in maintaining the genomic stability of mammalian cells and preventing malignant transformation. In this study, we investigated the intracellular diffusion of a p53-GFP fusion protein using confocal fluorescence recovery after photobleaching. We show that the diffusion of p53-GFP within the nucleus is well described by a mathematical model for diffusion of particles that bind temporarily to a spatially homogeneous immobile structure with binding and release rates k1 and k2, respectively. The diffusion constant of p53-GFP was estimated to be Dp53-GFP=15.4 microm2 s-1, significantly slower than that of GFP alone, DGFP=41.6 microm2 s-1. The reaction rates of the binding and unbinding of p53-GFP were estimated as k1=0.3 s-1 and k2=0.4 s-1, respectively, values suggestive of nonspecific binding. Consistent with this finding, the diffusional mobilities of tumor-derived sequence-specific DNA binding mutants of p53 were indistinguishable from that of the wild-type protein. These data are consistent with a model in which, under steady-state conditions, p53 is latent and continuously scans DNA, requiring activation for sequence-specific DNA binding.

Active Transport, Cell Nucleus↗
PubMed2006

Neoadjuvant concurrent paclitaxel and radiation in stage II/III breast cancer.

PURPOSE: The aim of this study was to determine the safety and pathologic response rates following neoadjuvant paclitaxel and radiation in patients with stage II/III breast cancer and to evaluate the use of sequential biopsies to allow an in vivo assessment of biological markers as potential predictive markers of response to this regimen. PATIENTS AND METHODS: Patients with high-risk, operable breast cancer were treated with three cycles of paclitaxel 175 mg/m2 every 3 weeks, followed by twice-weekly paclitaxel 30 mg/m2 and concurrent radiation. Core biopsies were obtained at baseline and 24 to 72 hours after the first cycle of paclitaxel. After completing neoadjuvant treatment, patients underwent definitive surgery. The primary end point was pathologic complete response, which is defined as the absence of any invasive cancer at surgery. Potential markers of therapeutic response were evaluated including markers of proliferation, apoptosis, p21, HER2, estrogen receptor, and progesterone receptor status. RESULTS: Of the 38 patients enrolled, 13 (34%) had a pathologic complete response. There was no significant difference in baseline Ki-67 between responders (35%) and nonresponders (28%; P = 0.45). There was also no significant change in Ki-67 following paclitaxel administration. Despite this lack of immunohistologic change in proliferative activity, baseline mitotic index was higher for patients with pathologic complete response over nonresponders (27 versus 10, P = 0.003). Moreover, the increase in mitotic index following paclitaxel administration was associated with pathologic complete response. CONCLUSIONS: Neoadjuvant paclitaxel/radiation is effective and well tolerated. Tumor proliferation at baseline and response to chemotherapy as measured by mitotic activity may serve as an important indicator of pathologic response to neoadjuvant paclitaxel/radiation.

Adult↗
PubMed2006

p63 and epithelial biology.

The transcription factor p63 is a homologue of the tumor suppressor p53. Unlike p53, which is dispensable for normal development, p63 is critical for the development of stratified epithelial tissues such as epidermis, breast, and prostate. p63 encodes multiple protein isoforms with both transactivating and transcriptional repressor activities that can regulate a wide spectrum of target genes. p63 is also implicated in tumor formation and progression in stratified epithelia, with evidence for both tumor suppressive and oncogenic properties. This review will examine current data and hypotheses regarding the role of p63 in the development, maintenance, and tumorigenesis of stratified epithelium.

Animals↗
PubMed2005

Ultraviolet radiation induces phosphorylation and ubiquitin-mediated degradation of DeltaNp63alpha.

DeltaNp63alpha, a homologue of the tumor suppressor p53, acts as a transcriptional repressor with dominant negative effects towards p53. Additionally, DeltaNp63alpha is overexpressed in a number of squamous cell carcinomas, suggesting a potential role in oncogenesis. However, the mechanisms regulating p63 have yet to be elucidated. The goal of the current study was to determine the effect of various genotoxic stresses on DeltaNp63alpha posttranslational modification and stability in normal and transformed squamous epithelial cells. We found that DeltaNp63alpha protein levels decreased after ultraviolet radiation and paclitaxel treatment of both normal and transformed cells. After UV and paclitaxel treatment, DeltaNp63alpha phosphorylation was significantly modulated. Additionally, DeltaNp63alpha protein levels were regulated in a proteasome-dependent manner in control and UV treated cells with increased DeltaNp63alpha ubiquitination after UV treatment or proteasome inhibition. Our studies provide insight to a mechanism for DeltaNp63alpha regulation during normal cell proliferation and, in particular, after stress. Further, the inverse regulation of p53 and DeltaNp63alpha protein levels after cell stress through opposing regulation of proteasome-mediated degradation may allow for rapid transcriptional changes of specific target genes that are consistent with the roles of these family members in tumor suppression and cell growth.

Cell Line↗
PubMed2005

Constitutively active K-cyclin/cdk6 kinase in Kaposi sarcoma-associated herpesvirus-infected cells.

BACKGROUND: Kaposi sarcoma-associated human herpesvirus (KSHV) encodes K-cyclin, a homologue of D-type cellular cyclins, which binds cyclin-dependent kinases to phosphorylate various substrates. K-cyclin/cdk phosphorylates a subset of substrates normally targeted by cyclins D, E, and A. We used cells naturally infected with KSHV to further characterize the biochemical features of K-cyclin. METHODS: We used immunoprecipitation with K-cyclin antibodies to examine the association of K-cyclin with cdk2, cdk6, p21Cip1, and p27Kip1 proteins in BC3 cells. We separated populations of BC3 cells enriched in cells in G1, S, or G2/M phases by elutriation and measured K-cyclin protein and the kinase activity of K-cyclin/cdk6 complexes. The half-life of K-cyclin and cyclin D2 proteins was determined by blocking protein synthesis with cycloheximide and measuring proteins in cell lysates by western blot analysis. We fused the entire K-cyclin sequence to the carboxyl-terminal sequence of cellular cyclin D that contains the PEST degradation sequence to produce K-cyclin/D2 and transfected K-cyclin/D2 into K-cyclin-negative cells to investigate the effect of the PEST sequence on K-cyclin's stability. RESULTS: Viral K-cyclin interacted with cyclin-dependent kinases cdk2, cdk4, and cdk6 and with the cyclin/cdk inhibitory proteins p21Cip1 and p27Kip1 in BC3 cell lysates. Unlike D-type cyclins, whose expression is cell cycle dependent, the level of K-cyclin was stable throughout the cell cycle, and the kinase associated with the K-cyclin/cdk6 complex was constitutively active. The half-life of K-cyclin (6.9 hours) was much longer than that of cellular cyclin D2 (0.6 hour) and that of K-cyclin/D2 (0.5 hour), probably because K-cyclin lacks the PEST degradation sequence present in D-type cyclins. CONCLUSION: The constitutive activation of K-cyclin/cdk complexes in KSHV-infected cells appears to result from the extended half-life of K-cyclin and may explain its role in Kaposi sarcoma.

Blotting, Western↗
PubMed2005

IGFBP-3 is a direct target of transcriptional regulation by DeltaNp63alpha in squamous epithelium.

DeltaNp63alpha is a nuclear transcription factor that maintains epithelial progenitor cell populations, is overexpressed in several epithelial cancers, and can negatively regulate apoptosis. However, the mechanisms by which DeltaNp63alpha promotes cell survival are unclear. DeltaNp63alpha has been reported to act as a transcriptional repressor, but specific target genes directly repressed by DeltaNp63alpha remain unidentified. Here, we present evidence that DeltaNp63alpha functions to negatively regulate the proapoptotic protein IGFBP-3. Disruption of p63 expression in squamous epithelial cells increases IGFBP-3 expression, whereas ectopic expression of DeltaNp63alpha down-regulates IGFBP-3. DeltaNp63alpha binds to sites in the IGFBP-3 gene in vivo and can modulate transcription through these sites. Furthermore, DeltaNp63alpha and IGFBP-3 expression patterns are inversely correlated in normal squamous epithelium and squamous cell carcinomas. These data suggest that IGFBP-3 is a target of transcriptional repression by DeltaNp63alpha and that this repression represents a mechanism by which tumors that overexpress p63 may be protected from apoptosis.

Apoptosis↗
PubMed2005

Chromatin immunoprecipitation-based screen to identify functional genomic binding sites for sequence-specific transactivators.

In various human diseases, altered gene expression patterns are often the result of deregulated gene-specific transcription factor activity. To further understand disease on a molecular basis, the comprehensive analysis of transcription factor signaling networks is required. We developed an experimental approach, combining chromatin immunoprecipitation (ChIP) with a yeast-based assay, to screen the genome for transcription factor binding sites that link to transcriptionally regulated target genes. We used the tumor suppressor p53 to demonstrate the effectiveness of the method. Using primary and immortalized, nontransformed cultures of human mammary epithelial cells, we isolated over 100 genomic DNA fragments that contain novel p53 binding sites. This approach led to the identification and validation of novel p53 target genes involved in diverse signaling pathways, including growth factor signaling, protein kinase/phosphatase signaling, and RNA binding. Our results yield a more complete understanding of p53-regulated signaling pathways, and this approach could be applied to any number of transcription factors to further elucidate complex transcriptional networks.

Binding Sites↗
PubMed2004

Head and neck cancer: meeting summary and research opportunities.

Head and neck squamous cell carcinoma (HNSCC) is the most common malignant neoplasm arising in the mucosa of the upper aerodigestive tract. Nearly two thirds of patients present with advanced (stage III and IV) disease. Fifty percent of HNSCC patients die of their disease, and 5% of HNSCC patients per year will develop additional second primary tumors. Currently used therapeutic modalities (surgery, radiation, and/or chemotherapy) have been associated with rather modest improvements in patient survival. The Head and Neck Cancer: Research and Therapeutic Opportunities Workshop (held in Washington, DC, May 24-26, 2004) was organized by the Division of Cancer Biology at the National Cancer Institute to identify research areas and directions that will advance understanding of HNSCC biology and accelerate clinical translation. The primary goal of the workshop was to identify the barriers that impede basic science discovery and the translation of these developments to the clinical setting. Over a 2.5-day period, experts in both HNSCC and other cancer-related fields met to identify and prioritize the key areas for future research. The overall consensus was that HNSCC is a relatively understudied malignancy and that investigations that focus on the biology of this tumor have the potential to impact significantly on the prevention and treatment of epithelial malignancies. The chief objective is to communicate these research goals to the cancer biology community and encourage more interest in HNSCC as a tumor model to test translational research hypotheses.

Carcinoma, Squamous Cell↗
PubMed2004

p63: Molecular complexity in development and cancer.

Discovery of the p53 homologs p63 and p73 has brought new excitement to the p53 field. Identification of homologous genes coding for several proteins with similar and antagonistic properties towards p53 has been both intriguing and perplexing. A multitude of properties have been attributed to these new homologs and this review will focus on the biochemical and biological aspects of one family member, p63. Although the most ancient member of the p53 family, p63 is the most recently discovered and the least is known about this family member. Unlike p53, whose protein expression is not readily detectable in epithelial cells unless they are exposed to various stress conditions, p63 is expressed in select epithelial cells at high levels under normal conditions. p63 is highly expressed in embryonic ectoderm and in the nuclei of basal regenerative cells of many epithelial tissues in the adult including skin, breast myoepithelium, oral epithelium, prostate and urothelia. In contrast to the tumor suppressive function of p53, over-expression of select p63 splice variants is observed in many squamous carcinomas suggesting that p63 may act as an oncogene. Undoubtedly, the biochemical and biological activities attributed to p63 over the next several years will be diverse and regulation of the p63 gene and its several protein products complex. The use of various model systems and the study of human disease should continue to lead to rapid advances in our understanding of the role of p63 in development, epithelial cell maintenance and tumorigenesis.

Animals↗
PubMed2004

Induction by transforming growth factor-beta1 of epithelial to mesenchymal transition is a rare event in vitro.

INTRODUCTION: Transforming growth factor (TGF)-beta1 is proposed to inhibit the growth of epithelial cells in early tumorigenesis, and to promote tumor cell motility and invasion in the later stages of carcinogenesis through the induction of an epithelial to mesenchymal transition (EMT). EMT is a multistep process that is characterized by changes in cell morphology and dissociation of cell-cell contacts. Although there is growing interest in TGF-beta1-mediated EMT, the phenotype is limited to only a few murine cell lines and mouse models. METHODS: To identify alternative cell systems in which to study TGF-beta1-induced EMT, 18 human and mouse established cell lines and cultures of two human primary epithelial cell types were screened for TGF-beta1-induced EMT by analysis of cell morphology, and localization of zonula occludens-1, E-cadherin, and F-actin. Sensitivity to TGF-beta1 was also determined by [3H]thymidine incorporation, flow cytometry, phosphorylation of Smad2, and total levels of Smad2 and Smad3 in these cell lines and in six additional cancer cell lines. RESULTS: TGF-beta1 inhibited the growth of most nontransformed cells screened, but many of the cancer cell lines were insensitive to the growth inhibitory effects of TGF-beta1. In contrast, TGF-beta1 induced Smad2 phosphorylation in the majority of cell lines, including cell lines resistant to TGF-beta1-mediated cell cycle arrest. Of the cell lines screened only two underwent TGF-beta1-induced EMT. CONCLUSION: The results presented herein show that, although many cancer cell lines have lost sensitivity to the growth inhibitory effect of TGF-beta1, most show evidence of TGF-beta1 signal transduction, but only a few cell lines undergo TGF-beta1-mediated EMT.

Actins↗
PubMed2004

Dominant negative p63 isoform expression in head and neck squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: p63, a member of the p53 family of genes, is vital for normal epithelial development and may play a critical role in epithelial tumor formation. Although p63 has been identified in various head and neck malignancies, a detailed analysis of which of the six isoforms of the p63 gene is present in normal mucosa and head and neck malignancies has not yet been performed. The study analyzed p63 isoform expression on the RNA and protein level in normal, diseased, and malignant mucosa of the head and neck to examine the differential expression of p63 isoforms in head and neck tumors versus adjacent nonmalignant tissue and to identify the predominant p63 isoform expressed in head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN: Three experiments were performed. In experiment 1, p63 expression was analyzed by immunohistochemical analysis in 36 HNSCC specimens and matched normal tissue control specimens harvested from the same patient. Western blot analysis was also performed on matched specimens to confirm the identity of the p63 isoforms that were found. In experiment 2, reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed on matched normal and tumor specimens to analyze and quantitatively compare p63 isoform expression at the RNA level. In experiment 3, p63 expression was evaluated by immunohistochemical analysis in oral lichen planus, a benign mucosal lesion marked by hyperdifferentiation and apoptosis. METHODS: Immunohistochemical analysis, RT-PCR, and Western blot analysis of p63 were performed on HNSCC specimens and matched normal tissue control specimens. p63 expression in oral lichen planus specimens was also examined by immunohistochemical analysis. RESULTS: In experiment 1, analysis of 36 HNSCC specimens from various head and neck subsites showed p63 expression in all tumors and matched normal tissue specimens (36 of 36). Western blot analyses indicated that dominant negative (DeltaN) isoform p63alpha (DeltaNp63alpha) is the major isoform expressed at the protein level in tumors and adjacent normal tissue. In experiment 2, RT-PCR analyses of 10 matched specimens confirmed that, although all three DeltaNp63 isoforms (DeltaNp63alpha, DeltaNp63beta, and DeltaNp63gamma) are expressed in normal and malignant mucosa of the head and neck, DeltaNp63alpha is the predominant transcript expressed. In experiment 3, immunohistochemical analysis of p63 in the pro-apoptotic condition of lichen planus indicated that p63 is underexpressed as compared with normal mucosal specimens. CONCLUSION: Although all three DeltaNp63 isoforms are present in HNSCC, DeltaNp63alpha protein is the predominant isoform expressed in these malignancies. DeltaNp63alpha is also overexpressed in tumors compared with matched normal tissue specimens and is underexpressed in the pro-apoptotic condition of lichen planus. These findings suggest that DeltaNp63alpha plays an anti-differentiation and anti-apoptotic role in the mucosal epithelium of the head and neck, possibly playing a pivotal role in the formation of HNSCC. Currently, DeltaNp63alpha is an attractive target for mechanistic study aimed at therapeutic intervention.

Base Sequence↗
PubMed2003

Significance of p63 amplification and overexpression in lung cancer development and prognosis.

The fight against lung cancer is greatly compromised by the lack of effective early detection strategies. Genomic abnormalities and specifically the amplification of chromosomal region 3q26-3qter in lung cancer represent a major signature of neoplastic transformation. Here, we address the significance of p53 homologue p63 mapping to 3q27 in lung tumorigenesis. We analyzed p63 gene copy number (CN) by fluorescence in situ hybridization and expression by immunohistochemistry in tissue microarrays of 217 non-small cell lung cancers (NSCLCs) and correlated them with survival. We additionally characterized our findings in a subset of 24 NSCLCs by reverse transcription-PCR and Western blotting. We analyzed p63 CN and protein expression in 41 preinvasive squamous lesions. The p63 genomic sequence was amplified in 88% of squamous carcinomas, in 42% of large cell carcinomas, and in 11% of adenocarcinomas of the lung. The predominant splice variant of p63 expressed was DeltaNp63alpha. Western analyses revealed DeltaNp63alpha expression in normal bronchus and squamous carcinomas but not in normal lung or in adenocarcinomas. Furthermore, p63genomic amplification and protein staining intensity associated with better survival. We found a significant increase in CN in preinvasive lesions graded severe dysplasia or higher. Our data demonstrate that there is early and frequent genomic amplification of p63 in the development of squamous carcinoma of the lung and that patients with NSCLC showing amplification and overexpression of p63 have prolonged survival. These observations suggest that p63 genomic amplification has an early role in lung tumorigenesis and deserves additional evaluation as a biomarker for lung cancer progression.

Alternative Splicing↗
PubMed2003

Delta Np63 alpha expression is regulated by the phosphoinositide 3-kinase pathway.

p63 is a homologue of p53 that functions to maintain progenitor cell populations in stratified epithelia. Delta Np63 alpha is overexpressed in epithelial cancers and has been shown to have oncogenic properties. We have previously reported that inhibition of epidermal growth factor receptor signaling results in a decrease in Delta Np63 alpha expression. Here, we demonstrate Delta Np63 alpha is a target of the phosphoinositide-3-kinase (PI3K) pathway downstream of the epidermal growth factor receptor. Treatment of keratinocytes with epidermal growth factor results in an increase in Delta Np63 alpha expression at the mRNA level, which is abrogated by inhibition of PI3K but not mitogen-activated protein kinase signaling. Small interfering RNA-mediated knockdown of the p110 beta catalytic subunit of PI3K results in a decrease in Delta Np63 alpha protein levels in keratinocytes. The results presented herein suggest that regulation of Delta Np63 alpha expression by the PI3K pathway plays a critical role in the survival and proliferative capacity of squamous epithelia.

Cell Line, Tumor↗
PubMed2003

Cell cycle-dependent transduction of cell-permeant Cre recombinase proteins.

Protein transduction has been widely used to analyze biochemical processes in living cells quantitatively and under non-steady-state conditions. The present study analyzed the effects of cell cycle on the uptake and activity of cell-permeant Cre recombinase proteins. Previous studies had suggested that the efficiency of recombination and/or protein transduction varied among individual cells, even within a clonal population. We report here that cells in the G1 phase of the cell cycle undergo recombination at a lower rate than cells at other phases of the cell cycle, and that this variation results largely from differences in protein uptake, associated with differences in cell size. These results have implications regarding the mechanism of protein transduction and identify a source of heterogeneity that can influence the response of individual cells to cell-permeant proteins.

Amino Acid Sequence↗
PubMed2003

Interaction of human NAD(P)H:quinone oxidoreductase 1 (NQO1) with the tumor suppressor protein p53 in cells and cell-free systems.

NAD(P)H:quinone oxidoreductase 1 (NQO1) has been proposed to stabilize p53 via a redox mechanism involving oxidation of NAD(P)H as a consequence of the catalytic activity of NQO1. We report that treatment of HCT-116 human colon carcinoma cells with the NQO1 inhibitor ES936 had no effect on the levels of p53 protein. ES936 is a mechanism-based inhibitor of NQO1 that irreversibly blocks the catalytic function of the enzyme. This suggests that a redox mechanism involving NQO1-mediated NAD(P)H oxidation is not responsible for the stabilization of p53. We also examined the ability of the NQO1 protein to associate with p53 using co-immunoprecipitation experiments. Results from these experiments demonstrated co-immunoprecipitation of NQO1 with p53 and vice versa. The association between p53 and NQO1 was not affected by treatment of HCT-116 cells with ES936, demonstrating that the association was not dependent on the catalytic activity of NQO1. A comparison of isogenic HCT-116 p53+/+ and HCT-116 p53-/- cells demonstrated an interaction of NQO1 and p53 only in the p53+/+ cells. Experiments performed in an in vitro transcription/translation system utilizing rabbit reticulocyte lysates confirmed the interaction of NQO1 and p53. In these experiments a full-length p53 coding region was used to express p53 in the presence of recombinant NQO1 protein. An association of p53 and NQO1 was also observed in primary human keratinocytes and mammary epithelial cells. In studies where mdm-2 co-immunoprecipitated with p53, no association of mdm-2 with NQO1 was observed. These data demonstrate an association between p53 and NQO1 that may represent an alternate mechanism of p53 stabilization by NQO1 in a wide variety of human cell types.

Cell-Free System↗
PubMed2003

Combined modality management of breast cancer: development of predictive markers through proteomics.

The treatment of localized breast cancer often requires the use of surgery, radiation, and chemotherapy. The optimal sequencing of these treatments remains controversial. Although randomized studies have not found an improvement in survival with the use of neoadjuvant chemotherapy, a higher percentage of patients can undergo breast conservation. This clinical setting also allows us the unique opportunity to measure in vivo response to a select drug or drug combination. While there have been many retrospective studies looking at prognostic markers of response, there have been very few prospective studies developing markers that predict ultimate outcome from a specific drug or drug combination. This review discusses some of the newer technologies being used to develop predictive markers of therapeutic response. Proteomics is the study of the complete set of proteins expressed in a cell. It is a powerful tool to compare two different samples. Serial biopsies that compare the protein profile before and 24 hours after a cycle of chemotherapy may allow us to determine particular protein profiles that predict for ultimate clinical outcome.

Antineoplastic Agents↗