Ruben Ramirez - Academia.edu (original) (raw)

Papers by Ruben Ramirez

Research paper thumbnail of Surface Analyses and Immune Reactivities of Major Cell Wall-Associated Proteins of Group A Streptococcus

Infection and Immunity, 2005

A proteomic analysis was undertaken to identify cell wall-associated proteins of Streptococcus py... more A proteomic analysis was undertaken to identify cell wall-associated proteins of Streptococcus pyogenes. Seventy-four distinct cell wall-associated proteins were identified, 66 of which were novel. Thirty-three proteins were immunoreactive with pooled S. pyogenes-reactive human antisera. Biotinylation of the GAS cell surface identified 23 cell wall-associated proteins that are surface exposed.

Research paper thumbnail of Eosinophils contribute to innate antiviral immunity and promote clearance of respiratory syncytial virus

Eosinophils are recruited to the lungs in response to respiratory syncytial virus (RSV) infection... more Eosinophils are recruited to the lungs in response to respiratory syncytial virus (RSV) infection; however, their role in promoting antiviral host defense remains unclear. Here, we demonstrate that eosinophils express TLRs that recognize viral nucleic acids, are activated and degranulate after singlestranded RNA (ssRNA) stimulation of the TLR-7-MyD88 pathway, and provide host defense against RSV that is MyD88 dependent. In contrast to wild-type mice, virus clearance from lung tissue was more rapid in hypereosinophilic (interleukin-5 trans-genic) mice. Transfer of wild-type but not MyD88-deficient eosinophils to the lungs of RSV-infected wild-type mice accelerated virus clearance and inhibited the development of airways hyperreactivity. Similar responses were observed when infected recipient mice were MyD88 deficient. Eosinophils isolated from infected hypereosinophilic MyD88-sufficient but not MyD88-deficient mice expressed greater amounts of IFN regulatory factor (IRF)-7 and eosinophil-associated ribonucleases EAR-1 and EAR-2. Hypereosinophilia in the airways of infected mice also correlated with increased expression of IRF-7, IFN-␤, and NOS-2, and inhibition of NO production with the NOS-2 inhibitor L-NMA partially reversed the accelerated virus clearance promoted by eosinophils. Collectively, our results demonstrate that eosinophils can protect against RSV in vivo, as they promote virus clearance and may thus limit virus-induced lung dysfunction. (Blood. 2007;110:1578-1586

Research paper thumbnail of Differences in ERK Activation in Squamous Mucosa in Patients Who Have Gastroesophageal Reflux Disease with and without Barrett's Esophagus

American Journal of Gastroenterology, 2005

In some patients with gastroesophageal reflux disease (GERD), the reflux-damaged esophageal squam... more In some patients with gastroesophageal reflux disease (GERD), the reflux-damaged esophageal squamous epithelium heals through the process of intestinal metaplasia (resulting in Barrett's esophagus) rather than through the regeneration of more squamous cells. We hypothesized that squamous epithelium in Barrett's esophagus might have abnormalities in activation of the extracellular-regulated kinases 1 and 2 (ERK1/2) signaling pathway that may facilitate esophageal repair through metaplasia in response to acid-induced injury. Endoscopic biopsies were taken from distal esophageal squamous mucosa in patients who had GERD with and without Barrett's esophagus and in controls, before and after esophageal perfusion with 0.1 N HCl acid. Basal ERK1/2 phosphorylation, acid-induced ERK1/2 activity and phosphorylation, and localization of phosphorylated ERK1/2 were determined using immunoblotting, Western blotting, and immunohistochemistry. Compared to patients with Barrett's esophagus, patients with GERD exhibited significantly lower baseline levels of phosphorylated ERK1/2 expression (35 +/- 4%vs 90 +/- 21% control, p= 0.01) Acid exposure significantly increased ERK1/2 activity (346.6 +/- 51.90 to 446.8 +/- 62.44 RIU, p= 0.02) and phosphorylation (3.55 +/- 1.26 to 4.49 +/- 1.25 [ratio phospho/total ERK], p= 0.01) in the squamous mucosa of GERD patients, but not in those with Barrett's esophagus or in controls. Between patients with Barrett's esophagus and patients with uncomplicated GERD, there are significant differences in baseline levels and in acid-induced activation of ERK1/2 in esophageal squamous epithelium. To our knowledge, this is the first description of a molecular, phenotypic feature that distinguishes the esophageal squamous mucosa of GERD patients with and without Barrett's esophagus.

Research paper thumbnail of Acid Has Antiproliferative Effects in Nonneoplastic Barrett's Epithelial Cells

American Journal of Gastroenterology, 2007

For patients with Barrett&amp... more For patients with Barrett's esophagus, physicians commonly prescribe antisecretory medications in dosages above those required to heal reflux esophagitis because acid has been shown to have proproliferative and antiapoptotic effects on Barrett's cancer cells and on Barrett's mucosal explants. For a number of reasons, these model systems may not be ideal for determining the effects of acid on benign Barrett's epithelial cells, however. We studied the effects of acid on proliferation and apoptosis in a nonneoplastic, telomerase-immortalized Barrett's epithelial cell line. Barrett's cells were treated with two 3-minute exposures to acidic media. Cell growth was determined using cell counts, proliferation was studied by flow cytometry, cell viability was determined by trypan blue staining, and apoptosis was assessed by TUNEL and Annexin V. The expression levels of p53 and p21 were determined by Western blotting. p53 siRNA was used to study the effect of p53 inhibition on total cell numbers after acid exposure. Acid exposure significantly decreased total cell numbers at 24 h without affecting either cell viability or apoptosis. Acid exposure resulted in cell cycle prolongation that was associated with greater expression of p53, but not p21. The acid-induced decrease in total cell numbers was abolished by p53 RNAi. Acid exposure has p53-mediated, antiproliferative effects in nonneoplastic Barrett's epithelial cells. These findings contradict the results of prior in vitro and ex vivo studies. We speculate that the prescription of antisecretory medications in dosages beyond those required to heal gastroesophageal reflux disease (GERD) symptoms and endoscopic signs could be detrimental. Controlled, prospective clinical trials are needed to determine the optimal level of acid suppression for patients with Barrett's esophagus.

Research paper thumbnail of GERD is associated with shortened telomeres in the squamous epithelium of the distal esophagus

American Journal of Physiology-gastrointestinal and Liver Physiology, 2007

Telomeres are repetitive DNA sequences located at the ends of chromosomes. Telomeres are shortene... more Telomeres are repetitive DNA sequences located at the ends of chromosomes. Telomeres are shortened by repeated cell divisions and by oxidative DNA damage, and cells with critically shortened telomeres cannot divide. We hypothesized that chronic, GERDinduced injury of the esophageal squamous epithelium results in progressive telomeric shortening that eventually might interfere with mucosal healing. To address our hypothesis, we compared telomere length and telomerase activity in biopsy specimens of esophageal squamous epithelium from GERD patients and control patients. Endoscopic biopsies were taken from the esophageal squamous epithelium of 38 patients with GERD [10 long-segment Barrett's esophagus (LSBE), 15 short-segment (SSBE), 13 GERD without Barrett's esophagus] and 16 control patients without GERD. Telomere length was assessed using the terminal restriction fragment assay, and telomerase activity was studied by the PCR-based, telomeric repeat amplification protocol assay. Patients with GERD had significantly shorter telomeres in the distal esophagus than controls [8.3±.5 vs 10.9±1.5 (SEM) Kbp, p=0.043]. Among the patients with GERD, telomere length in the distal esophagus did not differ significantly in those with and without Barrett's esophagus (LSBE 7.9±.8, SSBE 8.6±.9, GERD without BE 8.7±1.0 Kbp). No significant differences in telomerase activity in the distal esophagus were noted between patients with GERD and controls (4.0±.39 vs 5.2±.53 RIUs). Telomeres in the squamous epithelium of the distal esophagus of patients who have GERD, with and without Barrett's esophagus, are significantly shorter than those of patients without GERD despite similar levels of telomerase activity.

Research paper thumbnail of H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

PLOS One, 2009

Background: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, ... more Background: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent.

Research paper thumbnail of EGFR Signaling Is Required for TGF-b1-Mediated COX2 Induction in Human Bronchial Epithelial Cells

Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins and thromboxanes fro... more Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins and thromboxanes from free arachidonic acid. In- creasing evidence suggests that COX-2 plays a role in tumorigenesis. A variety of stimuli induce COX-2 and it is overexpressed in many tumors, including non-small cell lung cancer (NSCLC). We studied the regulation of COX-2 expression in immortalized human bron- chial epithelial

Research paper thumbnail of Increased expression and no mutation of the Flap endonuclease (FEN1) gene in human lung cancer

Oncogene, 2003

The underlying molecular mechanisms leading to microsatellite alteration and mutations in human l... more The underlying molecular mechanisms leading to microsatellite alteration and mutations in human lung cancer remain unknown. Since Flap endonuclease1 (Fen1), which functions in the base excision repair system, has been shown to be involved in tumor progression of mouse models with microsatellite instability in a haplo-insufficient manner, we performed expression and mutation analyses for FEN1 in human lung cancer cell lines. Reverse transcriptase PCR analysis revealed that all 49 lung cancer cell lines (20 small cell lung cancers (SCLCs) and 29 non-small cell lung cancers (NSCLCs)) expressed FEN1. In addition, microarray analysis showed that FEN1 expression was elevated significantly by 1.65-fold (P ¼ 0.001) in SCLC cell lines compared to normal lung controls (normal human lung cultures and immortalized normal human bronchial epithelial cell lines). FEN1 protein was abundantly expressed in all 23 lung cancer cell lines (10 SCLCs and 13 NSCLCs) and was expressed at lower levels in three of four normal lung epithelial culture controls. Direct sequencing of genomic DNAs revealed no FEN1 mutation in seven SCLCs and nine NSCLCs. As part of this analysis we discovered and sequenced a FEN1 pseudogene (GenBank accession #AY249897) located at 1p22.2. This pseudogene is amplified from cDNA preparations contaminated with genomic DNA and must be taken into account in any FEN1 mutation analysis studies. Our results suggest that alterations of FEN1 are not likely to contribute to development of lung cancer.

Research paper thumbnail of Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions

Genes & Development, 2001

Telomere shortening is the mechanism underlying replicative aging in fibroblasts. A variety of re... more Telomere shortening is the mechanism underlying replicative aging in fibroblasts. A variety of reports now claim that inactivation of the p16 INK4a /pRB pathway is required in addition to telomere maintenance for the immortalization of cells such as skin keratinocytes and breast epithelial cells. We here show that the premature growth arrest of these cell types can be explained by an inadequate culture environment. Providing mesenchymal/epithelial interactions by cultivating the telomerase-expressing cells on feeder layers avoids the growth arrest associated with increased p16 INK4a . These results do not support a telomere-independent mechanism of replicative aging. 3 Corresponding authors. E-MAIL Woodring.Wright@utsouthwestern.edu; FAX (214) 648-8694. E-MAIL Jerry.Shay@UTSouthwestern.edu; FAX (214) 648-8694. Article and publication are at www.genesdev.org/cgi/doi/10.1101/gad. 859201.

Research paper thumbnail of Telomerase Activity Concentrates in the Mitotically Active Segments of Human Hair Follicles

Journal of Investigative Dermatology, 1997

Research paper thumbnail of Mutation analysis of the coding sequences of MEK-1 and MEK-2 genes in human lung cancer cell lines

Research paper thumbnail of IL20, an anti-angiogenic cytokine that inhibits COX2 expression

Biochemical and Biophysical Research Communications, 2005

COX-2 overexpression and subsequent PGE 2 production are frequently associated with non-small cel... more COX-2 overexpression and subsequent PGE 2 production are frequently associated with non-small cell lung cancer and are implicated in tumor-mediated angiogenesis. Here, we report for the first time that IL-20 downregulates COX-2 and PGE 2 in human bronchial epithelial and endothelial cells. Flow cytometry analysis suggests that IL-20-dependent inhibition of COX-2/PGE 2 occurs through the IL-22R1/IL-20R2 dimers. In addition, we report that IL-20 exerts anti-angiogenic effects, inhibiting experimental angiogenesis. IL-20-mediated inhibition of PMA-induced angiogenesis occurs through the COX-2 regulatory pathway. Altogether our findings revealed that IL-20 is a negative modulator of COX-2/PGE 2 and inhibits angiogenesis.

Research paper thumbnail of A three-dimensional model of differentiation of immortalized human bronchial epithelial cells

Differentiation, 2006

A therapeutic approach being investigated for a variety of pathologies is tissue regeneration usi... more A therapeutic approach being investigated for a variety of pathologies is tissue regeneration using a patient's own cells. Such studies have been hampered due to the difficulty in growing epithelial cells for prolonged periods in culture. Replicative senescence due to short telomeres and p16 induced by culture stress work together to inhibit cell growth. Forced expression of telomerase (hTERT) can prevent replicative senescence, and expression of the cell cycle protein cdk4 can sequester p16, thereby immortalizing epithelial cells in culture. In the present study, we used this method to immortalize human bronchial epithelial cells (HBECs) to determine whether immortalized HBECs retain the ability to differentiate normally. HBECs were plated atop contracted collagen gels containing lung fibro-blasts. This three-dimensional (3D) tissue model was cultured initially submerged, then raised to the air/liquid interface for up to 28 days. Normal differentiation was assessed by the presence of ciliated cells, goblet (mucin-producing) cells, and basal epithelial cells. Scanning electron microscopic observations revealed both ciliated and non-ciliated cells in these 3D tissues. Histological examination revealed the presence of mucinproducing cells, and immunohistochemistry using antibodies against p63 and keratin 14 showed the presence of basal cells. These results demonstrate that immortalized HBECs retain the capacity to differentiate into each of three cell types: basal, mucin-producing, and columnar ciliated epithelial cells. Such cells will be useful cellular reagents for research in aging, cancer progression, as well as normal bronchial epithelial differentiation and will help progress the use of engineered cells to enhance tissue regeneration.

Research paper thumbnail of Different Roles for Caveolin-1 in the Development of Non-Small Cell Lung Cancer versus Small Cell Lung Cancer

Caveolin-1 (CAV1), an essential structural constituent of caveolae that plays an important role i... more Caveolin-1 (CAV1), an essential structural constituent of caveolae that plays an important role in cellular processes such as transport and signaling, has been implicated in the development of human cancers. However, it is unclear whether CAV1 is acting like an oncogene or tumor suppressor gene. We found that CAV1 expression was reduced or absent in 95% of small cell lung cancers (SCLCs; n ‫؍‬ 21 lines), whereas it was retained in 76% of non-small cell lung cancers (NSCLCs; n ‫؍‬ 25 lines) compared with normal human lung epithelial cultures, where it was abundantly expressed. CAV1 expression was tightly linked to the ability to grow attached to the plastic cell culture surface, whereas CAV1-nonexpressing lung cancers of both SCLC and NSCLC type grew as suspension cultures. In addition, attached lung cancer cultures expressed phosphorylated focal adhesion kinase, whereas suspension cultures did not. Lack of CAV1 expression was tightly associated with CAV1 promoter methylation (P < 0.0001) such that CAV1 methylation was found in 93% of SCLCs (n ‫؍‬ 15) and 9% of NSCLCs (n ‫؍‬ 11), whereas 5-aza-2deoxycytidine treatment restored CAV1 expression in SCLCs. Exogenous CAV1 expression in SCLCs significantly inhibited soft-agar colony formation but did not lead to attachment. By contrast, CAV1 knockdown in NSCLCs mediated by small interfering RNA against CAV1 led to inhibition of cellular proliferation and soft-agar and liquid colony formation. Importantly, CAV1 knockdown led to reduced phospho-focal adhesion kinase and RalA, but not RalB, levels in NSCLC cells. These results suggest different roles for CAV1 in SCLC, where CAV1 acts like a tumor suppressor gene, and NSCLC, where it appears required for survival and growth.

Research paper thumbnail of A Reproducible Laser-Wounded Skin Equivalent Model to Study the Effects of Aging In Vitro

Rejuvenation Research, 2004

Hypothesis: Skin aging involves both chronological and photoaging processes. The effects of these... more Hypothesis: Skin aging involves both chronological and photoaging processes. The effects of these processes are often overlapping and include changes in both the stratified epithelium and the fibroblast-rich dermis. Wound healing is frequently delayed and results in scarring. A skin equivalent model can be used to study the role of cells and the extracellular matrix in the process of wound healing. Current studies using this model employ a full-thickness wound placed atop a nonwounded dermis to mimic a partialthickness wound. However, a true reproducible partial-thickness wound model has yet to be described. In this study, we investigated whether a laser-wounded skin equivalent would be a useful partial-thickness wound healing model. Methods: Three lasers were compared for the ability to generate a reproducible wound: an erbium-YAG, a highpowered excimer, and a low-powered excimer laser. Reepithelialization ability was tested using newborn and adult skin keratinocytes, adult esophageal keratinocytes, and cdk4overexpressing newborn keratinocytes. Keratinocyte compartmentalization and basement membrane formation were assessed by immunofluorescence. Results: The erbium-YAG and high-powered excimer laser cut reproducible wounds but left the remaining surface either discolored due to thermal damage and/or ragged; keratinocytes were unable to migrate into the wound area. The low-powered excimer laser cut reproducible wounds, leaving the cut surface intact and visibly unaltered; keratinocytes reepithelialized the wound in a collagenase-dependent manner within 3 days; return of compartmentalization and basement membrane occurred within 14 days. Conclusion: The laser-wounded skin equivalent is an adjustable, reproducible partial-thickness wound model where keratinocyte Laser-wounded skin equivalent

Research paper thumbnail of Detection of Telomerase Activity in Malignant and Nonmalignant Skin Conditions

Journal of Investigative Dermatology, 1996

Research paper thumbnail of Expression of Nicotinic Acetylcholine Receptor Subunit Genes in Non-Small-Cell Lung Cancer Reveals Differences between Smokers and Nonsmokers

Research paper thumbnail of Progressive Increase in Telomerase Activity From Benign Melanocytic Conditions to Malignant Melanoma

Neoplasia, 1999

The expression of telomerase activity and the in situ localization of the human telomerase RNA co... more The expression of telomerase activity and the in situ localization of the human telomerase RNA component ( ) hTR in melanocytic skin lesions was evaluated in specimens from sixty-three patients. Specimens of melanocytic nevi, primary melanomas and subcutaneous metastases of melanoma were obtained from fifty-eight patients, whereas metastasized lymph nodes were obtained from five patients. Telomerase activity was determined in these specimens by using a Poly-( ) merase Chain Reaction-based assay TRAP . High relative mean telomerase activity levels were detected in ( metastatic melanoma subcutaneous metastasess 54.5, ) lymph node metastasess 56.5 . Much lower levels were detected in primary melanomas, which increased with ( advancing levels of tumor cell penetration Clark II s ) 0.02, Clark III s 1.1, and Clark IV s 1.9 . Twenty-six formalin-fixed, paraffin-embedded melanocytic lesions were sectioned and analyzed for telomerase RNA with a radioactive in situ hybridization assay. In situ hybridization studies with a probe to the template RNA component of telomerase confirmed that expression was almost exclusively confined to tumor cells and not infiltrating lymphocytes. These results indicate that levels of telomerase activity and telomerase RNA in melanocytic lesions correlate well with clinical stage and could potentially assist in the diagnosis of borderline lesions.

Research paper thumbnail of Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells

Oncogene, 2003

Many stimuli causing 'stress' or DNA damage in cells can produce phenotypes that overlap with tel... more Many stimuli causing 'stress' or DNA damage in cells can produce phenotypes that overlap with telomere-based replicative senescence. In epithelial systems, the p16/RB pathway may function as a stress senescence-signaling pathway independent of telomere shortening. Overexpressing cyclin-dependent kinase 4 (Cdk4) in human epidermal keratinocytes and human mammary epithelial cells not only prevents the p16 INK4a -associated premature growth arrest due to telomere-independent stress (e.g., inadequate culture conditions), but also bypasses the ensuing telomere-dependent senescence (M1). Overexpressed Cdk4 in epithelial cells induces a dramatic upregulation of p16 INK4a and milder upregulation of p53 and p21 WAF1 , which become unresponsive to UV irradiation. Despite the high levels of these checkpoint factors, Cdk4-overexpressing cells divide in an apparently normal regulated fashion, are able to respond to changes in calcium levels, retain the stem cell phenotype, and fully differentiate and stratify. These results suggest that the differentiation pathways in Cdk4overexpressing cells remain intact.

Research paper thumbnail of ANALISIS CUENCA TEUSACA TRABAJO FINAL

Las tres partes del área del globo terrestre están conformadas por agua, de ella depende la vida ... more Las tres partes del área del globo terrestre están conformadas por agua, de ella depende la vida tanto para humanos como para el resto de los seres vivientes animales, plantas, etc. En el estudio de la tierra se examinan los fenómenos naturales aplicando procesos físicos. De tal manera se analiza la distribución del agua en la tierra, sus reacciones físicas y químicas con otras sustancias existentes en la naturaleza.

Research paper thumbnail of Surface Analyses and Immune Reactivities of Major Cell Wall-Associated Proteins of Group A Streptococcus

Infection and Immunity, 2005

A proteomic analysis was undertaken to identify cell wall-associated proteins of Streptococcus py... more A proteomic analysis was undertaken to identify cell wall-associated proteins of Streptococcus pyogenes. Seventy-four distinct cell wall-associated proteins were identified, 66 of which were novel. Thirty-three proteins were immunoreactive with pooled S. pyogenes-reactive human antisera. Biotinylation of the GAS cell surface identified 23 cell wall-associated proteins that are surface exposed.

Research paper thumbnail of Eosinophils contribute to innate antiviral immunity and promote clearance of respiratory syncytial virus

Eosinophils are recruited to the lungs in response to respiratory syncytial virus (RSV) infection... more Eosinophils are recruited to the lungs in response to respiratory syncytial virus (RSV) infection; however, their role in promoting antiviral host defense remains unclear. Here, we demonstrate that eosinophils express TLRs that recognize viral nucleic acids, are activated and degranulate after singlestranded RNA (ssRNA) stimulation of the TLR-7-MyD88 pathway, and provide host defense against RSV that is MyD88 dependent. In contrast to wild-type mice, virus clearance from lung tissue was more rapid in hypereosinophilic (interleukin-5 trans-genic) mice. Transfer of wild-type but not MyD88-deficient eosinophils to the lungs of RSV-infected wild-type mice accelerated virus clearance and inhibited the development of airways hyperreactivity. Similar responses were observed when infected recipient mice were MyD88 deficient. Eosinophils isolated from infected hypereosinophilic MyD88-sufficient but not MyD88-deficient mice expressed greater amounts of IFN regulatory factor (IRF)-7 and eosinophil-associated ribonucleases EAR-1 and EAR-2. Hypereosinophilia in the airways of infected mice also correlated with increased expression of IRF-7, IFN-␤, and NOS-2, and inhibition of NO production with the NOS-2 inhibitor L-NMA partially reversed the accelerated virus clearance promoted by eosinophils. Collectively, our results demonstrate that eosinophils can protect against RSV in vivo, as they promote virus clearance and may thus limit virus-induced lung dysfunction. (Blood. 2007;110:1578-1586

Research paper thumbnail of Differences in ERK Activation in Squamous Mucosa in Patients Who Have Gastroesophageal Reflux Disease with and without Barrett's Esophagus

American Journal of Gastroenterology, 2005

In some patients with gastroesophageal reflux disease (GERD), the reflux-damaged esophageal squam... more In some patients with gastroesophageal reflux disease (GERD), the reflux-damaged esophageal squamous epithelium heals through the process of intestinal metaplasia (resulting in Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s esophagus) rather than through the regeneration of more squamous cells. We hypothesized that squamous epithelium in Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s esophagus might have abnormalities in activation of the extracellular-regulated kinases 1 and 2 (ERK1/2) signaling pathway that may facilitate esophageal repair through metaplasia in response to acid-induced injury. Endoscopic biopsies were taken from distal esophageal squamous mucosa in patients who had GERD with and without Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s esophagus and in controls, before and after esophageal perfusion with 0.1 N HCl acid. Basal ERK1/2 phosphorylation, acid-induced ERK1/2 activity and phosphorylation, and localization of phosphorylated ERK1/2 were determined using immunoblotting, Western blotting, and immunohistochemistry. Compared to patients with Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s esophagus, patients with GERD exhibited significantly lower baseline levels of phosphorylated ERK1/2 expression (35 +/- 4%vs 90 +/- 21% control, p= 0.01) Acid exposure significantly increased ERK1/2 activity (346.6 +/- 51.90 to 446.8 +/- 62.44 RIU, p= 0.02) and phosphorylation (3.55 +/- 1.26 to 4.49 +/- 1.25 [ratio phospho/total ERK], p= 0.01) in the squamous mucosa of GERD patients, but not in those with Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s esophagus or in controls. Between patients with Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s esophagus and patients with uncomplicated GERD, there are significant differences in baseline levels and in acid-induced activation of ERK1/2 in esophageal squamous epithelium. To our knowledge, this is the first description of a molecular, phenotypic feature that distinguishes the esophageal squamous mucosa of GERD patients with and without Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s esophagus.

Research paper thumbnail of Acid Has Antiproliferative Effects in Nonneoplastic Barrett's Epithelial Cells

American Journal of Gastroenterology, 2007

For patients with Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp... more For patients with Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s esophagus, physicians commonly prescribe antisecretory medications in dosages above those required to heal reflux esophagitis because acid has been shown to have proproliferative and antiapoptotic effects on Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s cancer cells and on Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s mucosal explants. For a number of reasons, these model systems may not be ideal for determining the effects of acid on benign Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s epithelial cells, however. We studied the effects of acid on proliferation and apoptosis in a nonneoplastic, telomerase-immortalized Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s epithelial cell line. Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s cells were treated with two 3-minute exposures to acidic media. Cell growth was determined using cell counts, proliferation was studied by flow cytometry, cell viability was determined by trypan blue staining, and apoptosis was assessed by TUNEL and Annexin V. The expression levels of p53 and p21 were determined by Western blotting. p53 siRNA was used to study the effect of p53 inhibition on total cell numbers after acid exposure. Acid exposure significantly decreased total cell numbers at 24 h without affecting either cell viability or apoptosis. Acid exposure resulted in cell cycle prolongation that was associated with greater expression of p53, but not p21. The acid-induced decrease in total cell numbers was abolished by p53 RNAi. Acid exposure has p53-mediated, antiproliferative effects in nonneoplastic Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s epithelial cells. These findings contradict the results of prior in vitro and ex vivo studies. We speculate that the prescription of antisecretory medications in dosages beyond those required to heal gastroesophageal reflux disease (GERD) symptoms and endoscopic signs could be detrimental. Controlled, prospective clinical trials are needed to determine the optimal level of acid suppression for patients with Barrett&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s esophagus.

Research paper thumbnail of GERD is associated with shortened telomeres in the squamous epithelium of the distal esophagus

American Journal of Physiology-gastrointestinal and Liver Physiology, 2007

Telomeres are repetitive DNA sequences located at the ends of chromosomes. Telomeres are shortene... more Telomeres are repetitive DNA sequences located at the ends of chromosomes. Telomeres are shortened by repeated cell divisions and by oxidative DNA damage, and cells with critically shortened telomeres cannot divide. We hypothesized that chronic, GERDinduced injury of the esophageal squamous epithelium results in progressive telomeric shortening that eventually might interfere with mucosal healing. To address our hypothesis, we compared telomere length and telomerase activity in biopsy specimens of esophageal squamous epithelium from GERD patients and control patients. Endoscopic biopsies were taken from the esophageal squamous epithelium of 38 patients with GERD [10 long-segment Barrett's esophagus (LSBE), 15 short-segment (SSBE), 13 GERD without Barrett's esophagus] and 16 control patients without GERD. Telomere length was assessed using the terminal restriction fragment assay, and telomerase activity was studied by the PCR-based, telomeric repeat amplification protocol assay. Patients with GERD had significantly shorter telomeres in the distal esophagus than controls [8.3±.5 vs 10.9±1.5 (SEM) Kbp, p=0.043]. Among the patients with GERD, telomere length in the distal esophagus did not differ significantly in those with and without Barrett's esophagus (LSBE 7.9±.8, SSBE 8.6±.9, GERD without BE 8.7±1.0 Kbp). No significant differences in telomerase activity in the distal esophagus were noted between patients with GERD and controls (4.0±.39 vs 5.2±.53 RIUs). Telomeres in the squamous epithelium of the distal esophagus of patients who have GERD, with and without Barrett's esophagus, are significantly shorter than those of patients without GERD despite similar levels of telomerase activity.

Research paper thumbnail of H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

PLOS One, 2009

Background: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, ... more Background: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent.

Research paper thumbnail of EGFR Signaling Is Required for TGF-b1-Mediated COX2 Induction in Human Bronchial Epithelial Cells

Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins and thromboxanes fro... more Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins and thromboxanes from free arachidonic acid. In- creasing evidence suggests that COX-2 plays a role in tumorigenesis. A variety of stimuli induce COX-2 and it is overexpressed in many tumors, including non-small cell lung cancer (NSCLC). We studied the regulation of COX-2 expression in immortalized human bron- chial epithelial

Research paper thumbnail of Increased expression and no mutation of the Flap endonuclease (FEN1) gene in human lung cancer

Oncogene, 2003

The underlying molecular mechanisms leading to microsatellite alteration and mutations in human l... more The underlying molecular mechanisms leading to microsatellite alteration and mutations in human lung cancer remain unknown. Since Flap endonuclease1 (Fen1), which functions in the base excision repair system, has been shown to be involved in tumor progression of mouse models with microsatellite instability in a haplo-insufficient manner, we performed expression and mutation analyses for FEN1 in human lung cancer cell lines. Reverse transcriptase PCR analysis revealed that all 49 lung cancer cell lines (20 small cell lung cancers (SCLCs) and 29 non-small cell lung cancers (NSCLCs)) expressed FEN1. In addition, microarray analysis showed that FEN1 expression was elevated significantly by 1.65-fold (P ¼ 0.001) in SCLC cell lines compared to normal lung controls (normal human lung cultures and immortalized normal human bronchial epithelial cell lines). FEN1 protein was abundantly expressed in all 23 lung cancer cell lines (10 SCLCs and 13 NSCLCs) and was expressed at lower levels in three of four normal lung epithelial culture controls. Direct sequencing of genomic DNAs revealed no FEN1 mutation in seven SCLCs and nine NSCLCs. As part of this analysis we discovered and sequenced a FEN1 pseudogene (GenBank accession #AY249897) located at 1p22.2. This pseudogene is amplified from cDNA preparations contaminated with genomic DNA and must be taken into account in any FEN1 mutation analysis studies. Our results suggest that alterations of FEN1 are not likely to contribute to development of lung cancer.

Research paper thumbnail of Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions

Genes & Development, 2001

Telomere shortening is the mechanism underlying replicative aging in fibroblasts. A variety of re... more Telomere shortening is the mechanism underlying replicative aging in fibroblasts. A variety of reports now claim that inactivation of the p16 INK4a /pRB pathway is required in addition to telomere maintenance for the immortalization of cells such as skin keratinocytes and breast epithelial cells. We here show that the premature growth arrest of these cell types can be explained by an inadequate culture environment. Providing mesenchymal/epithelial interactions by cultivating the telomerase-expressing cells on feeder layers avoids the growth arrest associated with increased p16 INK4a . These results do not support a telomere-independent mechanism of replicative aging. 3 Corresponding authors. E-MAIL Woodring.Wright@utsouthwestern.edu; FAX (214) 648-8694. E-MAIL Jerry.Shay@UTSouthwestern.edu; FAX (214) 648-8694. Article and publication are at www.genesdev.org/cgi/doi/10.1101/gad. 859201.

Research paper thumbnail of Telomerase Activity Concentrates in the Mitotically Active Segments of Human Hair Follicles

Journal of Investigative Dermatology, 1997

Research paper thumbnail of Mutation analysis of the coding sequences of MEK-1 and MEK-2 genes in human lung cancer cell lines

Research paper thumbnail of IL20, an anti-angiogenic cytokine that inhibits COX2 expression

Biochemical and Biophysical Research Communications, 2005

COX-2 overexpression and subsequent PGE 2 production are frequently associated with non-small cel... more COX-2 overexpression and subsequent PGE 2 production are frequently associated with non-small cell lung cancer and are implicated in tumor-mediated angiogenesis. Here, we report for the first time that IL-20 downregulates COX-2 and PGE 2 in human bronchial epithelial and endothelial cells. Flow cytometry analysis suggests that IL-20-dependent inhibition of COX-2/PGE 2 occurs through the IL-22R1/IL-20R2 dimers. In addition, we report that IL-20 exerts anti-angiogenic effects, inhibiting experimental angiogenesis. IL-20-mediated inhibition of PMA-induced angiogenesis occurs through the COX-2 regulatory pathway. Altogether our findings revealed that IL-20 is a negative modulator of COX-2/PGE 2 and inhibits angiogenesis.

Research paper thumbnail of A three-dimensional model of differentiation of immortalized human bronchial epithelial cells

Differentiation, 2006

A therapeutic approach being investigated for a variety of pathologies is tissue regeneration usi... more A therapeutic approach being investigated for a variety of pathologies is tissue regeneration using a patient's own cells. Such studies have been hampered due to the difficulty in growing epithelial cells for prolonged periods in culture. Replicative senescence due to short telomeres and p16 induced by culture stress work together to inhibit cell growth. Forced expression of telomerase (hTERT) can prevent replicative senescence, and expression of the cell cycle protein cdk4 can sequester p16, thereby immortalizing epithelial cells in culture. In the present study, we used this method to immortalize human bronchial epithelial cells (HBECs) to determine whether immortalized HBECs retain the ability to differentiate normally. HBECs were plated atop contracted collagen gels containing lung fibro-blasts. This three-dimensional (3D) tissue model was cultured initially submerged, then raised to the air/liquid interface for up to 28 days. Normal differentiation was assessed by the presence of ciliated cells, goblet (mucin-producing) cells, and basal epithelial cells. Scanning electron microscopic observations revealed both ciliated and non-ciliated cells in these 3D tissues. Histological examination revealed the presence of mucinproducing cells, and immunohistochemistry using antibodies against p63 and keratin 14 showed the presence of basal cells. These results demonstrate that immortalized HBECs retain the capacity to differentiate into each of three cell types: basal, mucin-producing, and columnar ciliated epithelial cells. Such cells will be useful cellular reagents for research in aging, cancer progression, as well as normal bronchial epithelial differentiation and will help progress the use of engineered cells to enhance tissue regeneration.

Research paper thumbnail of Different Roles for Caveolin-1 in the Development of Non-Small Cell Lung Cancer versus Small Cell Lung Cancer

Caveolin-1 (CAV1), an essential structural constituent of caveolae that plays an important role i... more Caveolin-1 (CAV1), an essential structural constituent of caveolae that plays an important role in cellular processes such as transport and signaling, has been implicated in the development of human cancers. However, it is unclear whether CAV1 is acting like an oncogene or tumor suppressor gene. We found that CAV1 expression was reduced or absent in 95% of small cell lung cancers (SCLCs; n ‫؍‬ 21 lines), whereas it was retained in 76% of non-small cell lung cancers (NSCLCs; n ‫؍‬ 25 lines) compared with normal human lung epithelial cultures, where it was abundantly expressed. CAV1 expression was tightly linked to the ability to grow attached to the plastic cell culture surface, whereas CAV1-nonexpressing lung cancers of both SCLC and NSCLC type grew as suspension cultures. In addition, attached lung cancer cultures expressed phosphorylated focal adhesion kinase, whereas suspension cultures did not. Lack of CAV1 expression was tightly associated with CAV1 promoter methylation (P < 0.0001) such that CAV1 methylation was found in 93% of SCLCs (n ‫؍‬ 15) and 9% of NSCLCs (n ‫؍‬ 11), whereas 5-aza-2deoxycytidine treatment restored CAV1 expression in SCLCs. Exogenous CAV1 expression in SCLCs significantly inhibited soft-agar colony formation but did not lead to attachment. By contrast, CAV1 knockdown in NSCLCs mediated by small interfering RNA against CAV1 led to inhibition of cellular proliferation and soft-agar and liquid colony formation. Importantly, CAV1 knockdown led to reduced phospho-focal adhesion kinase and RalA, but not RalB, levels in NSCLC cells. These results suggest different roles for CAV1 in SCLC, where CAV1 acts like a tumor suppressor gene, and NSCLC, where it appears required for survival and growth.

Research paper thumbnail of A Reproducible Laser-Wounded Skin Equivalent Model to Study the Effects of Aging In Vitro

Rejuvenation Research, 2004

Hypothesis: Skin aging involves both chronological and photoaging processes. The effects of these... more Hypothesis: Skin aging involves both chronological and photoaging processes. The effects of these processes are often overlapping and include changes in both the stratified epithelium and the fibroblast-rich dermis. Wound healing is frequently delayed and results in scarring. A skin equivalent model can be used to study the role of cells and the extracellular matrix in the process of wound healing. Current studies using this model employ a full-thickness wound placed atop a nonwounded dermis to mimic a partialthickness wound. However, a true reproducible partial-thickness wound model has yet to be described. In this study, we investigated whether a laser-wounded skin equivalent would be a useful partial-thickness wound healing model. Methods: Three lasers were compared for the ability to generate a reproducible wound: an erbium-YAG, a highpowered excimer, and a low-powered excimer laser. Reepithelialization ability was tested using newborn and adult skin keratinocytes, adult esophageal keratinocytes, and cdk4overexpressing newborn keratinocytes. Keratinocyte compartmentalization and basement membrane formation were assessed by immunofluorescence. Results: The erbium-YAG and high-powered excimer laser cut reproducible wounds but left the remaining surface either discolored due to thermal damage and/or ragged; keratinocytes were unable to migrate into the wound area. The low-powered excimer laser cut reproducible wounds, leaving the cut surface intact and visibly unaltered; keratinocytes reepithelialized the wound in a collagenase-dependent manner within 3 days; return of compartmentalization and basement membrane occurred within 14 days. Conclusion: The laser-wounded skin equivalent is an adjustable, reproducible partial-thickness wound model where keratinocyte Laser-wounded skin equivalent

Research paper thumbnail of Detection of Telomerase Activity in Malignant and Nonmalignant Skin Conditions

Journal of Investigative Dermatology, 1996

Research paper thumbnail of Expression of Nicotinic Acetylcholine Receptor Subunit Genes in Non-Small-Cell Lung Cancer Reveals Differences between Smokers and Nonsmokers

Research paper thumbnail of Progressive Increase in Telomerase Activity From Benign Melanocytic Conditions to Malignant Melanoma

Neoplasia, 1999

The expression of telomerase activity and the in situ localization of the human telomerase RNA co... more The expression of telomerase activity and the in situ localization of the human telomerase RNA component ( ) hTR in melanocytic skin lesions was evaluated in specimens from sixty-three patients. Specimens of melanocytic nevi, primary melanomas and subcutaneous metastases of melanoma were obtained from fifty-eight patients, whereas metastasized lymph nodes were obtained from five patients. Telomerase activity was determined in these specimens by using a Poly-( ) merase Chain Reaction-based assay TRAP . High relative mean telomerase activity levels were detected in ( metastatic melanoma subcutaneous metastasess 54.5, ) lymph node metastasess 56.5 . Much lower levels were detected in primary melanomas, which increased with ( advancing levels of tumor cell penetration Clark II s ) 0.02, Clark III s 1.1, and Clark IV s 1.9 . Twenty-six formalin-fixed, paraffin-embedded melanocytic lesions were sectioned and analyzed for telomerase RNA with a radioactive in situ hybridization assay. In situ hybridization studies with a probe to the template RNA component of telomerase confirmed that expression was almost exclusively confined to tumor cells and not infiltrating lymphocytes. These results indicate that levels of telomerase activity and telomerase RNA in melanocytic lesions correlate well with clinical stage and could potentially assist in the diagnosis of borderline lesions.

Research paper thumbnail of Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells

Oncogene, 2003

Many stimuli causing 'stress' or DNA damage in cells can produce phenotypes that overlap with tel... more Many stimuli causing 'stress' or DNA damage in cells can produce phenotypes that overlap with telomere-based replicative senescence. In epithelial systems, the p16/RB pathway may function as a stress senescence-signaling pathway independent of telomere shortening. Overexpressing cyclin-dependent kinase 4 (Cdk4) in human epidermal keratinocytes and human mammary epithelial cells not only prevents the p16 INK4a -associated premature growth arrest due to telomere-independent stress (e.g., inadequate culture conditions), but also bypasses the ensuing telomere-dependent senescence (M1). Overexpressed Cdk4 in epithelial cells induces a dramatic upregulation of p16 INK4a and milder upregulation of p53 and p21 WAF1 , which become unresponsive to UV irradiation. Despite the high levels of these checkpoint factors, Cdk4-overexpressing cells divide in an apparently normal regulated fashion, are able to respond to changes in calcium levels, retain the stem cell phenotype, and fully differentiate and stratify. These results suggest that the differentiation pathways in Cdk4overexpressing cells remain intact.

Research paper thumbnail of ANALISIS CUENCA TEUSACA TRABAJO FINAL

Las tres partes del área del globo terrestre están conformadas por agua, de ella depende la vida ... more Las tres partes del área del globo terrestre están conformadas por agua, de ella depende la vida tanto para humanos como para el resto de los seres vivientes animales, plantas, etc. En el estudio de la tierra se examinan los fenómenos naturales aplicando procesos físicos. De tal manera se analiza la distribución del agua en la tierra, sus reacciones físicas y químicas con otras sustancias existentes en la naturaleza.