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A K Rustgi

Publications and source records attributed to A K Rustgi.

At least 37 records · Page 2Linked to original sources
PubMed2000

Dual function of the epithelial specific ets transcription factor, ELF3, in modulating differentiation.

The ets family of transcription factors comprises many members which contribute to diverse cellular functions that vary depending upon the cell- and tissue-type context. Recently, different groups have identified a novel member of the ets family that is epithelial-specific. Variably called ESE-1, ERT, jen, ESX, this gene is designated currently as ELF3. In order to understand transcriptional regulatory mechanisms mediated by ELF3, we investigated its effect on the human keratin 4 gene promoter based upon the role of keratin 4 in early differentiation of the esophageal squamous epithelium. Interestingly, ELF3 suppressed basal keratin 4 promoter activity in both esophageal and cervical epithelial cancer cell lines, a novel result, while simultaneously activating the late-differentiation linked SPRR2A promoter. Furthermore, serial deletion constructs of the keratin 4 promoter continued to be suppressed by ELF3, a phenomenon that was only partially rescued by ELF3 ets domain mutants, but completely abrogated by deletion of the ELF3 pointed domain. These results suggest that ELF3 may have dual functions in the transcriptional regulation of genes involved in squamous epithelial differentiation. One of these functions may not be exclusively mediated through DNA binding in the context of transcriptional suppression of the keratin 4 promoter.

Amino Acid Sequence↗
PubMed2000

Intestinal cell kinase (ICK) localizes to the crypt region and requires a dual phosphorylation site found in map kinases.

Identification of key regulatory kinases in the intestinal epithelium are useful to understand the molecular mechanisms that underlie proliferation and differentiation in cells found in this compartment. We used the polymerase chain reaction (PCR) to amplify the catalytic kinase domain of serine-threonine kinases by employing degenerate primers and then screened an intestinal crypt cDNA library to clone and sequence the open reading frame of a novel serine-threonine kinase. This was then further characterized by Northern blot analysis and RNA in situ hybridization. This kinase, designated intestinal cell kinase, harbors a dual phosphorylation site found in mitogen-activating protein (MAP) kinases that is important for kinase activity.

Amino Acid Sequence↗
PubMed2000

Gastroesophageal reflux disease and Barrett's esophagus.

GERD has emerged as an important medical issue by virtue not only of its high prevalence, but also by the concern that it may predispose to adenocarcinoma of the esophagus. It generally is classified into erosive and nonerosive forms. Nonerosive GERD tends to remain as such in most patients, and treatment is based on symptom relief. In contrast, erosive GERD mandates aggressive lifelong treatment based on its inevitable relapse without appropriate treatment. Two excellent therapies are available for GERD. Proton-pump inhibitors are highly efficacious and have an excellent long-term (> 10 years) safety profile. Laparoscopic fundoplication offers a new and easier method of delivering a standard reliable procedure also with excellent long-term efficacy. The occurrence of Barrett's esophagus among reflux patients has emerged as an important problem mostly because of the rapidly rising incidence of adenocarcinoma of the esophagus in the population. All patients with long-standing reflux symptoms should be screened for Barrett's and subsequently followed regularly with surveillance endoscopy if Barrett's is detected. Although there are no data to show that aggressive medical or surgical treatment alters the malignant potential of this disease, patients need lifelong therapy.

Adenocarcinoma↗
PubMed1999

Cyclin D1 overexpression combined with N-nitrosomethylbenzylamine increases dysplasia and cellular proliferation in murine esophageal squamous epithelium.

We previously described the oral-esophageal tissue-specific expression of cyclin D1 with the Epstein-Barr virus ED-L2 promoter in transgenic mice, and resulting dysplasia. Given the evidence for an interplay between environmental and genetic factors in esophageal squamous carcinogenesis, the aim of this study was to determine the potential cooperation of the nitrosamine compound N-nitrosomethylbenzylamine (NMBA), an esophageal specific carcinogen, in the cyclin D1 transgenic mice. NMBA was first demonstrated to induce dysplasia in two strains of inbred mice, C57BL/6 and FVB/N. Subcutaneous NMBA was then administrated to wild type and transgenic mice beginning at 4 weeks of age. Mice were monitored for the duration of the study for general appearance, activity and weight, and were euthanized at 12 and 15 months. Histopathologic analysis revealed increased severity of dysplasia in cyclin D1 mice treated with NMBA compared with treated age-matched wild-type mice and untreated mice. There was also increased proliferating cell nuclear antigen (PCNA) expression in the esophagi of NMBA treated cyclin D1 mice. Taken together, these findings suggest that a genetic alteration, specifically cyclin D1 overexpression and a chemical carinogen, NMBA, may cooperate to increase the severity of esophageal squamous dysplasia, a prominent precursor to carcinoma.

Animals↗
PubMed1999

Mapping of a target region of allelic loss to a 0.5-cM interval on chromosome 22q13 in human colorectal cancer.

BACKGROUND & AIMS: Chromosomal allelic losses have a varying frequency in colorectal cancer. The aim of this study was to define the target region of allelic loss on chromosome 22q in human colorectal carcinogenesis. METHODS: Fifty-seven pairs of matched normal colonic mucosa and tumor specimens from patients with colorectal cancer, as well as 15 colon cancer-derived cell lines, were genotyped using 15 microsatellite markers spanning chromosome 22q. A potential candidate gene was analyzed by a single-strand conformation polymorphism (SSCP)/direct DNA sequencing approach. RESULTS: After excluding 7 tumors with evidence of microsatellite instability, allelic loss was observed in 11 of the informative tumors (22%), 5 of which exhibited losses in all informative loci. The remaining 6 tumors showed variable patterns of partial allelic loss on chromosome 22q, thereby localizing a minimal region of allelic deletion between markers D22S1171 and D22S928. Physical mapping showed that this interval was 0.57 cM consisting of approximately 425 kilobases. Database searches identified the NBK/BIK gene, a proapoptotic BCL-2 family member, as a candidate gene in that region. However, SSCP/sequencing analysis excluded mutations of this gene. CONCLUSIONS: This study provides evidence for the involvement of putative tumor-suppressor gene(s) on chromosome 22q in human colorectal carcinogenesis. The identification of a 0.5-cM interval serves as the basis for the isolation of such a gene by positional cloning.

Alleles↗
PubMed1998

Mouse keratin 4 is necessary for internal epithelial integrity.

Keratins are intermediate filaments of epithelial cells. Mutations in keratin genes expressed in skin lead to human disorders, including epidermolysis bullosa simplex and epidermolytic hyperkeratosis. We examined the role of keratin 4 (K4) in maintaining the integrity of internal epithelial linings by using gene targeting to generate mice containing a null mutation in the epithelial K4 gene. Homozygous mice that do not express K4 develop a spectrum of phenotypes that affect several organs which express K4 including the esophagus, tongue, and cornea. The cellular phenotypes include basal hyperplasia, lack of maturation, hyperkeratosis, atypical nuclei, perinuclear clearing, and cell degeneration. These results are consistent with the notion that K4 is required for internal epithelial cell integrity. As mutations in K4 in humans lead to a disorder called white sponge nevus, the K4-deficient mice may serve as models for white sponge nevus and for understanding the role of K4 in cellular proliferation and differentiation.

Aging↗
PubMed1998

Transcriptional regulation of the differentiation-linked human K4 promoter is dependent upon esophageal-specific nuclear factors.

The stratified squamous epithelium comprises actively proliferating basal cells that undergo a program of differentiation accompanied by morphological, biochemical, and genetic changes. The transcriptional regulatory signals and the genes that orchestrate this switch from proliferation to differentiation can be studied through the keratin gene family. Given the localization of keratin 4 (K4) to the early differentiated suprabasal compartment and having previously demonstrated that targeted disruption of this gene in murine embryonic stem cells results in impairment of the normal differentiation program in esophageal and corneal epithelial cells, we studied the transcriptional regulation of the human K4 promoter. A panel of K4 promoter deletions were found in transient transfection assays to be predominantly active in esophageal and corneal cell lines. A critical cis-regulatory element resides between -163 and -140 bp and contains an inverted CACACCT motif. A site-directed mutated version of this motif within the K4 promoter renders it inactive, whereas the wild-type version is active in a heterologous promoter system. It specifically binds esophageal-specific zinc-dependent transcriptional factors. Our studies demonstrate that regulation of the human K4 promoter is in part mediated through tissue-specific transcriptional factors.

Animals↗
PubMed1998

Transactivation of the human keratin 4 and Epstein-Barr virus ED-L2 promoters by gut-enriched Krüppel-like factor.

The Krüppel-like family of transcription factors comprises genes that appear to have tissue-restricted functions. Expression of gut-enriched Krüppel-like factor (GKLF) may be important in the switch from proliferation to differentiation in the squamous epithelium. We sought to determine transcriptionally mediated effects of GKLF on two promoters active in the esophageal squamous epithelium, namely the Epstein-Barr virus ED-L2 and human keratin 4 promoters. Both promoters contain a CACCC-like motif previously shown to bind GKLF. To determine whether GKLF regulates genes containing this element, we first demonstrated expression and then cloned the full-length human GKLF from an esophageal squamous carcinoma cell line. In a transient transfection system, GKLF increased the activity of both promoters >25-fold, localized to regions containing the CACCC-like element. Recombinant GKLF specifically binds the CACCC-like motif in both promoters. GKLF epitope-tagged protein leads to the formation of two proteins of 65 and 34 kDa. The chromatographically purified 65-kDa protein binds the CACCC-like element from both Epstein-Barr virus ED-L2 and keratin 4 promoters, which is not attenuated by the 34-kDa protein. In summary, GKLF is expressed in esophageal squamous epithelial cells and transcriptionally activates two esophageal epithelial promoters important at the transition toward differentiation.

Cell Line↗
PubMed1998

Cellular characterization and successful transfection of serially subcultured normal human esophageal keratinocytes.

BACKGROUND: In vitro cell culture models can provide unique insights into squamous epithelial proliferation, differentiation, and neoplastic transformation. Cultures of human esophageal keratinocytes could be advantageous for the study of these processes. METHODS: Normal human esophageal keratinocytes were cultivated on 3T3 fibroblast feeder layers in vitro and expanded through four serial subcultivations. Confluent tertiary cultures were analyzed by morphological and immunohistochemical techniques to define their basic properties. The ability to transiently transfect cultured esophageal epithelium was tested using a Rous sarcoma virus-luciferase reporter gene by the calcium phosphate and lipofection methods. RESULTS: Postconfluent cultures displayed a predominantly basal cell phenotype with limited stratification, widespread expression of keratins 5 and 14, and production of attachment specialization proteins such as alpha6beta4 integrin and collagen VII. Terminal differentiation markers (involucrin and transglutaminase) were prematurely expressed. The cells expressed growth factors important in proliferation and differentiation, such as transforming growth factor-beta and interleukin-1beta. Tertiary cultures were successfully transiently transfected with a Rous sarcoma virus-luciferase reporter gene construct. CONCLUSION: Normal human esophageal cells can be serially passaged through extended numbers of cell generations and transfected by standard methods. This in vitro system may be useful in the study of fundamental cellular processes governing proliferation and differentiation in the esophageal epithelium.

3T3 Cells↗
PubMed1998

Dose perturbation caused by high-density inhomogeneities in small beams in stereotactic radiosurgery.

The influence of high-density tissue heterogeneities in small-diameter beams used in stereotactic radiosurgery has been investigated. Dose perturbation immediately behind aluminium sheets, used to simulate a high-density tissue inhomogeneity such as bone, was studied in a solid water phantom. Dose reduction factors (DRFs), which are the ratios of the dose in the presence of the inhomogeneity to dose in a uniform density solid water phantom, were measured with a diamond detector for three thicknesses of aluminium. DRFs exhibit dependence on both the inhomogeneity thickness and the beam diameter. The DRF decreases with inhomogeneity thickness. The DRF initially decreases with increase in the beam diameter from 12.5 to 25 mm. For fields greater than 25 mm, the DRFs are nearly constant. The commonly used algorithms such as the TAR ratio method underestimate the magnitude of the measured effect. A good agreement between these measurements and Monte Carlo calculations is obtained. The influence of the high-density inhomogeneity on the tissue maximum ratio (TMR) was also measured with the inhomogeneity at a fixed depth dmax from the entrance surface. The TMR is reduced for all detector-inhomogeneity distances investigated. The dose build-up phenomenon observed in the presence of low-density air inhomogeneity is absent in the presence of a high-density inhomogeneity. The beam width (defined by 50% dose points) immediately beyond the inhomogeneity is unaffected by the high-density inhomogeneity. However, the 90%-10% and 80%-20% dose penumbra widths and the dose outside the beam edge (beyond the 50% dose point) are reduced. This reduction in dose outside the beam edge is caused by the reduced range of the secondary radiation (photons and electrons) in the high-density medium.

Data Interpretation, Statistical↗
PubMed1997

Transforming growth factor-alpha enhances cyclin D1 transcription through the binding of early growth response protein to a cis-regulatory element in the cyclin D1 promoter.

Cyclin D1 is a critical oncogene involved in the regulation of progression through the G1 phase of the cell cycle, thereby contributing to cell proliferation. This is mediated through interaction of cyclin D1 with its catalytic partners, the cyclin-dependent kinases, and the subsequent phosphorylation of the retinoblastoma protein. Cyclin D1, in turn, is regulated by mitogenic stimuli. We demonstrate that transforming growth factor-alpha (TGFalpha) induces cyclin D1 mRNA in esophageal squamous epithelial cells, and this appears to correlate with increased cyclin D1 protein expression and cyclin-dependent kinase 6 activity. The induction of cyclin D1 transcription by TGFalpha is mediated in part through the induction of the early growth response protein (Egr-1) and its subsequent binding of Egr-1 to a cis-regulatory region spanning nucleotides -144 to -104 of the cyclin D1 promoter. The Egr-1 binding activity to the cyclin D1 promoter appears to require de novo protein synthesis and is not influenced by Sp1 binding to overlapping Sp1 motifs. Taken together, these data provide evidence that TGFalpha enhances cyclin D1 transcription through the induction of Egr-1 binding to a cis-regulatory region in the cyclin D1 promoter. This has important mechanistic implications into the transcriptional regulation of cyclin D1 by an essential proproliferative growth factor and cell cycle progression.

Cyclin D1↗
PubMed1997

A transgenic mouse model with cyclin D1 overexpression results in cell cycle, epidermal growth factor receptor, and p53 abnormalities.

The cyclin D1 oncogene is critical in the progression of the cell cycle through the G1 phase. It is frequently overexpressed in squamous cell carcinomas originating from the head/neck and esophagus. Yet, the functional consequences of aberrant cyclin D1 overexpression are not entirely understood apart from increased cell proliferation. To address this question, we have developed a transgenic mouse model in which the EBV ED-L2 promoter targets cyclin D1 to the stratified squamous epithelium in a tissue-specific fashion to the tongue and esophagus, thereby resulting in a dysplastic phenotype. We now demonstrate that the dysplastic phenotype is associated with increased cell proliferation based on proliferating cell nuclear antigen overexpression and abnormalities in cyclin-dependent kinase 4, epidermal growth factor receptor, and p53. In aggregate, these studies suggest that alterations in certain oncogenes and tumor suppressor genes occur early during head/neck and esophageal carcinogenesis.

Animals↗
PubMed1997

The keratinocyte-specific Epstein-Barr virus ED-L2 promoter is regulated by phorbol 12-myristate 13-acetate through two cis-regulatory elements containing E-box and Krüppel-like factor motifs.

We previously employed 782 base pairs of the Epstein-Barr virus ED-L2 early lytic cycle promoter in a transgenic mouse model to target cyclin D1 to the stratified squamous epithelium of the tongue and esophagus. This promoter is located 5' to the transcriptional start site of a short open reading frame BNLF-2A and is immediately 3' to the BNLF-1 (LMP-1 oncogene) open reading frame. We studied transcriptional regulation of the ED-L2 promoter by phorbol 12-myristate 13-acetate (PMA) as a means of understanding the tissue specificity of this promoter. The transcriptional activity of the ED-L2 promoter was stimulated 40-fold by PMA and could be blocked with the compound H7 through antagonism of protein kinase C. 5' deletion analysis of the 782-base pair promoter demonstrated that the sequences necessary for PMA-stimulated trans-activation were located in two separate cis-regulatory regions of the promoter: -187 to -164 and -144 to -114 base pairs from the transcription start site of BNLF-2A. Importantly, mutation of critical base pairs in each region was sufficient to abolish PMA-stimulated trans-activation in the native ED-L2 promoter. Region -187 to -164 contains a CACCTG (E-box) motif, and region -144 to -114 contains a CACACCC motif. Both of these motifs are necessary for trans-activation by PMA. These regions do not, however, demonstrate enhancer characteristics when tested in a heterologous minimal promoter system. Variations of the CACACCC motif are found in other keratinocyte-specific promoters, as well as in the DNA binding motifs of the Krüppel-like family of transcription factors. Electrophoretic mobility shift assays with specific competitors and factor-specific antibody supershift assays demonstrated that one complex binding the -187 to -164 region containing the CACCTG nucleotides has characteristics of the helix-loop-helix protein upstream stimulatory factor, whereas a factor binding the CACACCC motif may be a member of the Krüppel-like family. These experiments show how ubiquitous and tissue-specific transcription factors induced by PMA regulate the ED-L2 promoter in squamous epithelial cells.

Cell Lineage↗
PubMed1997

A CACCC box-like cis-regulatory element of the Epstein-Barr virus ED-L2 promoter interacts with a novel transcriptional factor in tissue-specific squamous epithelia.

The Epstein-Barr (EBV) virus induces a lytic state after infecting epithelial cells. Subsequently, there is infection of B lymphocytes with two types of cycles, latent and lytic. Apart from linkage of the EBV latent membrane protein-1 (LMP-1) with benign and malignant conditions of squamous epithelial cells, little is known about other EBV gene products that may be important in these processes as well as cellular transcriptional factors that regulate EBV gene expression in these epithelial cells. The EBV ED-L2 promoter, an early lytic cycle promoter, is located upstream of a transcription start site for a short open reading frame designated BNLF2 and just downstream of the BNLF1 (LMP-1) open reading frame. We have previously used the EBV ED-L2 promoter to target oncogenes in transgenic mice, resulting in tissue-specific expression in the tongue, esophagus, forestomach, and skin, all sharing stratifying squamous epithelia, alternatively called keratinocytes. In the present study, we have functionally dissected the ED-L2 promoter by making deletion constructs fused to the luciferase reporter gene with transient transfections into squamous and nonsquamous epithelial cell lines as well as B lymphocytes. A CACCC box-like cis-regulatory element has been identified that is located between -218 and -187 base pairs of the ED-L2 promoter that confers significant promoter activity only in squamous epithelial cells. This cis-regulatory element is active in a heterologous minimal herpes simplex virus thymidine kinase promoter reporter gene construct when transfected into squamous epithelial cells but not in nonsquamous epithelial cells. DNA gel mobility shift assays have led to the identification of DNA-protein complexes that bind the CACCC box-like element. One of these proteins is a novel transcriptional factor that is uniquely active in stratified squamous epithelial cells, designated as keratinocyte specific factor (KSF). KSF may be related to Sp1 but appears to be distinct from Sp1. In addition, KSF may interact with related or identical cis-regulatory elements found in human papillomavirus-11 E6 and cytokeratin K3 promoters that are active in keratinocytes. In aggregate, KSF may be important in the transcriptional regulation of viral and eukaryotic genes in keratinocytes.

Animals↗