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Biomedical subjects

A K Rustgi

Publications and source records attributed to A K Rustgi.

At least 19 recordsLinked to original sources
PubMed2006

AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.

Epidermal growth factor receptor (EGFR) overexpression and activation is critical in the initiation and progression of cancers, especially those of epithelial origin. EGFR activation is associated with the induction of divergent signal transduction pathways and a gamut of cellular processes; however, the cell-type and tissue-type specificity conferred by certain pathways remains to be elucidated. In the context of the esophageal epithelium, a prototype stratified squamous epithelium, EGFR overexpression is relevant in the earliest events of carcinogenesis as modeled in a three-dimensional organotypic culture system. We demonstrate that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, and not the MEK/MAPK (mitogen-activated protein kinase) pathway, is preferentially activated in EGFR-mediated esophageal epithelial hyperplasia, a premalignant lesion. The hyperplasia was abolished with direct inhibition of PI3K and of AKT but not with inhibition of the MAPK pathway. With the introduction of an inducible AKT vector in both primary and immortalized esophageal epithelial cells, we find that AKT overexpression and activation is permissive for complete epithelial formation in organotypic culture, but imposes a growth constraint in cells grown in monolayer. In organotypic culture, AKT mediates changes related to cell shape and size with an expansion of the differentiated compartment.

Cell Differentiation↗
PubMed2004

Recruitment of patients into an internet-based clinical trials database: the experience of OncoLink and the National Colorectal Cancer Research Alliance.

PURPOSE: In March 2001, the National Colorectal Cancer Research Alliance (NCCRA) and OncoLink (http://www.oncolink.org) established a database to facilitate patient enrollment onto clinical trials. This study describes the population registering with the database and identifies discrepancies between individuals registering through the Internet and those registering through a telephone call center. METHODS: Participants registered with the NCCRA/OncoLink database through the Internet or a telephone call center. All participants entering the database completed a questionnaire regarding basic demographics, colon cancer risk factors, and indicated how they became aware of the database. Comparisons were made between individuals registering through the Internet and those registering through the telephone call center. RESULTS: A total of 2,162 participants registered during the first 16 months of the database. Most patients registered through the Internet rather than the telephone call center (88% v 12%; P < .001). More females than males registered (73% v 27%; P < .001). The majority (89%) were white. Participants registering through the Internet were younger than those registering through the call center (mean, 48.8 v 55.0 years; P < .001). There was no difference between the two groups with regard to sex or ethnicity. CONCLUSION: The Internet has the potential to increase the likelihood that interested individuals find appropriate clinical trials. Some of the discrepancies that are known to exist for access to the Internet were also seen for those registering with the database through the Internet. Despite these differences, the potential to increase clinical trial enrollment with this type of Internet-based database is high.

Adolescent↗
PubMed2002

Cell cycle dysregulation in oral cancer.

The dysregulation of the molecular events governing cell cycle control is emerging as a central theme of oral carcinogenesis. Regulatory pathways responding to extracellular signaling or intracellular stress and DNA damage converge on the cell cycle apparatus. Abrogation of mitogenic and anti-mitogenic response regulatory proteins, such as the retinoblastoma tumor suppressor protein (pRB), cyclin D1, cyclin-dependent kinase (CDK) 6, and CDK inhibitors (p21(WAF1/CIP1), p27(KIP1), and p16(INK4a)), occur frequently in human oral cancers. Cellular responses to metabolic stress or genomic damage through p53 and related pathways that block cell cycle progression are also altered during oral carcinogenesis. In addition, new pathways and cell cycle regulatory proteins, such as p12(DOC-1), are being discovered. The multistep process of oral carcinogenesis likely involves functional alteration of cell cycle regulatory members combined with escape from cellular senescence and apoptotic signaling pathways. Detailing the molecular alterations and understanding the functional consequences of the dysregulation of the cell cycle apparatus in the malignant oral keratinocyte will uncover novel diagnostic and therapeutic approaches.

Alphapapillomavirus↗
PubMed2001

p63 expression is associated with p53 loss in oral-esophageal epithelia of p53-deficient mice.

The p53 gene family, comprising p53, p63, and p73, has overlapping and distinctive functional roles. These members share structural similarities allowing for dynamic interplay in the activation of genes that are important in development and key cellular functions, such as the induction of apoptosis. Whereas p53 is a classical tumor suppressor gene, p63 and p73 do not share this feature in cancer formation and progression. The compensation in the expression level of these members in a background that is deficient for one of them has not been examined previously. Given the importance of p63 in the development and differentiation of oral-esophageal stratified squamous epithelia and the absence of oral-esophageal tumors in p53-null mice, we postulated and describe herein that p63 expression is associated with the loss of p53 in a p53-deficient background. Both full-length and amino-truncated forms of p63 are expressed and increased in oral-esophageal epithelia of p53-null mice when compared with wild-type mice, and the induction of p21 may potentially be preserved through the increase of p63.

Animals↗
PubMed2001

Paclitaxel induces prolonged activation of the Ras/MEK/ERK pathway independently of activating the programmed cell death machinery.

Paclitaxel is a widely used chemotherapeutic agent and is known to induce programmed cell death (apoptosis) in a variety of cell types, but the precise underlying mechanisms are poorly understood. To elucidate these mechanisms, we challenged human esophageal squamous cancer cell lines with paclitaxel and investigated its effects upon signal transduction pathways. Physiologically relevant concentrations of paclitaxel (1-1,000 nm) induced apoptosis. All three mitogen-activated protein kinase (MAPK) family members, c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK) were activated upon paclitaxel treatment. Interestingly, JNK activation and p38 MAPK activation were delayed and peaked at 48 h, whereas ERK activity was sustained over 72 h. In addition, Ras activation and MAPK/ERK kinase (MEK) phosphorylation were observed in concordance with ERK activation. While ERK activation was completely ablated by MEK inhibitors, immunoprecipitation and Western blot analysis revealed that neither MEK-1 nor MEK-2 was involved, but instead another member of the MEK family may potentially participate. Although pretreatment with a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone rescued the cell death, it did not prevent Ras or ERK activation. Furthermore, inhibition of JNK, p38 MAPK, or MEK did not alter PARP cleavage and the cell death induced by paclitaxel. These results in aggregate suggest that the delayed activation of JNK, p38 MAPK, and ERK was not linked to activation of the cell death machinery.

Amino Acid Chloromethyl Ketones↗
PubMed2001

The keratin 19 promoter is potent for cell-specific targeting of genes in transgenic mice.

BACKGROUND & AIMS: Keratins are intermediate filaments that are critical in cytoskeletal organization. Their roles in cellular processes are underscored by inherited human diseases in which germline mutations of keratins are found, as well as by transgenic and knockout mouse models that recapitulate those diseases. Keratin 19 (K19) has unique structural properties and developmental and spatial expression patterns. This suggests that K19 expression may correlate with important cell fate decisions in gastrointestinal tract epithelia. METHODS: We used mouse K19 5' untranslated region and promoter sequences and fused it to the lacZ reporter gene in a transgene construct. Characterization was by beta-galactosidase expression and X-gal histochemistry in gastrointestinal epithelia. Because endogenous K19 protein is transcriptionally regulated by the Kruppel-like transcription factor 4 (KLF4), we determined the spatial expression patterns of KLF4 and K19 in relationship to the lacZ reporter gene product. RESULTS: K19-lacZ transgenic mice were found to have reporter gene expression in an epithelial-specific pattern. Expression was restricted to ductal epithelial cells in the pancreas, surface colonocytes, small intestinal villi, and gastric isthmus cells. Transgene expression correlated with K19 and KLF4 protein expression in the pancreas and stomach and was overlapping in the small and large intestine. CONCLUSIONS: The K19 promoter may be a useful tool to study epithelial cell biology and subsequent transdifferentiation programs, particularly the pancreas and stomach.

Animals↗
PubMed2001

Cyclin D1 overexpression and p53 inactivation immortalize primary oral keratinocytes by a telomerase-independent mechanism.

The immortalization of human cells is a critical step in multistep carcinogenesis. Oral-esophageal carcinomas, a model system to investigate molecular mechanisms underlying squamous carcinogenesis, frequently involve cyclin D1 overexpression and inactivation of the p53 tumor suppressor. Therefore, our goal was to establish the functional role of cyclin D1 overexpression and p53 inactivation in the immortalization of primary human oral squamous epithelial cells (keratinocytes) as an important step toward malignant transformation. Cyclin D1 overexpression alone was found to induce extension of the replicative life span of normal oral keratinocytes, whereas the combination of cyclin D1 overexpression and p53 inactivation led to their immortalization. This study also demonstrates that immortalization of oral keratinocytes can be independent of telomerase activation, involving an alternative pathway of telomere maintenance (ALT).

Carcinoma, Squamous Cell↗
PubMed2000

Akt/protein kinase B isoforms are differentially regulated by epidermal growth factor stimulation.

Overexpression of epidermal growth factor receptor (EGFR) in certain cancers is well established. There is growing evidence that epidermal growth factor (EGF) activates Akt/protein kinase B (PKB) in a phosphoinositide 3-OH kinase (PI3K)-dependent manner, but it is not yet clear which Akt isoforms are involved in this signal transduction pathway. We investigated the functional regulation of three Akt isoforms, Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma, in esophageal cancer cells where EGFR is frequently overexpressed. Upon EGF simulation, phosphorylation of Akt1 at the Ser-473 residue was remarkably induced. This result was corroborated by in vitro Akt kinase assays using glycogen synthase kinase 3beta as the substrate. PI3K inhibitors, wortmannin or LY294002, significantly blocked the Akt kinase activity induced by EGF. Akt2 activity was evaluated by electrophoretic mobility shift assays. Robust activation of Akt2 by EGF was observed in some cell lines in a PI3K-dependent manner. EGF-induced Akt3 activation was demonstrated by Ser-472 phosphorylation of Akt3 but in a restrictive fashion. In aggregate, EGF-mediated activation of Akt isoforms is overlapping and distinctive. The mechanism by which EGFR recruits the PI3K/Akt pathway was in part differentially regulated at the level of Ras but independent of heterodimerization of EGFR with either ErbB2 or ErbB3 based upon functional dissection of pathways in esophageal cancer cell lines.

Androstadienes↗
PubMed2000

The tissue-dependent keratin 19 gene transcription is regulated by GKLF/KLF4 and Sp1.

Keratins play critical roles in cellular differentiation and cytoskeletal organization. Keratin 19 (K19) is unique because it has been implicated as a marker of stem cells in some tissues, such as the hair follicle in the skin. It is also associated with malignant transformation in esophageal and pancreatic cancers. Here, we show that the K19 promoter is active in a subset of gastrointestinal cancer cells derived from esophageal and pancreas but inactive in other contexts. This activity was mapped to a short region containing an overlapping binding site for gut-enriched Krüppel-like factor (GKLF/KLF4) and Sp1. GKLF has a higher binding affinity and is the predominant binding factor in cells with low Sp-1 protein levels. Pancreatic acinar cells normally do not express K19, but overexpression of GKLF and Sp1 in these cells leads to aberrant expression, similar to what is observed in pancreatic cancer. These results demonstrate the functional interaction of ubiquitous and tissue-restricted transcription factors in determining tissue- and neoplasm-specific patterns of gene expression.

3T3 Cells↗
PubMed2000

Ultrastructural and ERG findings in mice with adenomatous polyposis coli gene disruption.

PURPOSE: In order to continue the previous morphological studies of eyes from mice with adenomatous polyposis coli (APC) gene mutation at codon 1638, we determined the ultrastructural and electrophysiologic characteristics of these eyes. METHODS: Thirty-eight eyes from 20 mice heterozygous for APC gene mutation and 22 eyes from 11 wild-type mice were examined by light microscopy. Six APC-modified eyes without light microscopic abnormalities, four APC-modified eyes with focal light microscopic abnormalities, and four wild-type eyes were examined by electron microscopy. Electroretinograms were recorded from four APC-modified and three wild-type mice. RESULTS: Four of 38 APC-modified eyes demonstrated ultrastructural evidence of focal RPE cells with increased melanosome production and atrophy. Other areas of the RPE in these four eyes demonstrated no ultrastructural abnormalities. Three APC-modified eyes demonstrated electron and light microscopic evidence of RPE hyperplasia. Electron microscopic examination of APC-modified eyes without light microscopic evidence of abnormalities demonstrated no ultrastructural differences from age-matched controls. Electroretinography demonstrated no differences in the b-wave or c-wave amplitudes between APC-modified and wild-type mice. CONCLUSIONS: While light microscopic RPE alterations are observed in these APC-modified mice, the absence of a generalized, ultrastructural murine RPE defect is in contradistinction to observations in electron microscopic investigations of humans with colonic polyposis, pigmented ocular fundus lesions, and APC gene mutations between codons 463 and 1444. Our results in mice with APC mutation at codon 1638, however, are consistent with a previously identified association between the expression of pigmented ocular fundus lesions and region-specific mutation in the human APC gene. The APC protein may possess a physiologic function for both retinal and RPE development.

Adenomatous Polyposis Coli↗
PubMed2000

Interaction between Sp1 and cell cycle regulatory proteins is important in transactivation of a differentiation-related gene.

The stratified squamous epithelium is a model system in which to define molecular mechanisms underlying the switch from proliferation to differentiation. This can be achieved through the functional dissection of keratin gene promoters. Having previously established the importance of keratin 4 in maintaining the differentiated phenotype in corneal epithelial cells, we investigated the role of Sp1-mediated transactivation of the keratin 4 promoter given the role of Sp1 in differentiation and cell cycle progression. Sp1 transactivation of the keratin 4 promoter was diminished in cyclin D1-overexpressing cells, which may be mediated through a newly described direct interaction between Sp1 and cyclin D1 and opposed by a complex between Sp1 and pRB.

Animals↗
PubMed2000

A region of deletion on chromosome 22q13 is common to human breast and colorectal cancers.

Chromosomal allelic losses have varying frequency in breast cancer, with key regions including chromosomes 1, 3p, 7q, 9p, 16q, 17, and 22q. Recently, we have been able to map a new target region of allelic loss on chromosome 22q involved in colorectal cancer. The aim of the current investigation was to determine whether this target region may also be involved in human breast carcinogenesis. Thirty-six pairs of matched normal and tumor specimens from breast cancer patients, as well as eight breast cancer-derived cell lines, were genotyped using 17 microsatellite markers spanning chromosome 22q. Allelic deletion was found in 19 of 36 tumors (53%), and the pattern observed in those cases with partial losses was consistent with a region flanked by D22S1171 and D22S928. This interval overlaps that identified in colorectal cancer and comprises nearly 1.1 Mb. This study provides evidence of a common region of deletion on chromosome 22q13 involved in both breast and colorectal cancers and underscores the existence of putative tumor suppressor gene(s) at this location.

Alleles↗
PubMed2000

The Krüppel-like transcriptional factors Zf9 and GKLF coactivate the human keratin 4 promoter and physically interact.

Zf9/CPBP/KLF6 is a widely expressed member of the Krüppel-like family of transcriptional factors which regulates gene expression in hepatic stellate cells. Because of its ubiquitous expression including in the esophagus, we have explored its function in the esophageal squamous epithelium, a model system to study cellular proliferation and differentiation. Reverse transcription-PCR (RT-PCR) and Western blot analyses revealed that Zf9 was highly expressed in human esophageal squamous cancer cell lines. Additionally, Zf9 localizes to the esophageal squamous epithelium by immunohistochemistry. Using transient transfection, Zf9 transactivates the human keratin 4 (K4) promoter reporter gene construct in a subset of the esophageal cancer cell lines through indirect mechanisms. Co-transfection of Zf9 and GKLF/KLF4, which is also a member of the Krüppel-like factors and expressed in the esophageal squamous epithelium, leads to coactivation in an additive fashion. Furthermore, we demonstrate that there is a physical interaction between GKLF and Zf9, a novel finding for Krüppel-like family members.

Cell Line↗