Biomedical subjects

C L Walker

Publications and source records attributed to C L Walker.

At least 37 records · Page 2Linked to original sources

PubMed2000

Application of high-performance liquid chromatography-based analysis of DNA fragments to molecular carcinogenesis.

Denaturing high-performance liquid chromatography (DHPLC)-based DNA fragment analysis is a high-throughput technology that can be used to obtain information on both genetic alterations and gene expression. By using different approaches based on polymerase chain reaction, this technique can be used to determine loss or gain of an allele, to quantitate the amount of RNA expressed, and to detect a single nucleotide change. Applications of DHPLC to molecular carcinogenesis include genotyping of transgenic animals; determination of allelic imbalances, including loss of heterozygosity in tumors; measurement of changes in gene expression; and detection of DNA polymorphisms and point mutations. In our laboratories DHPLC has been validated and used to genotype an Eker rat colony, to study the genetic profile of renal cell carcinomas, to quantitate expression of the keratinocyte lipid-binding protein gene in 8-lipoxygenase transgenic mice, and to detect polymorphisms and a point mutation in the tuberous sclerosis 2 tumor suppressor gene in t-haplotype mice.

Animals↗

PubMed1999

Estrogen receptor activation via activation function 2 predicts agonism of xenoestrogens in normal and neoplastic cells of the uterine myometrium.

The possible contribution of endocrine disrupters to human disease, particularly those compounds that modulate the estrogen receptor (ER), has recently drawn considerable attention. The tissue specificity of effects mediated by the ER is well recognized, although the mechanism of this specificity is not understood sufficiently to predict the effects of a particular ligand in different target tissues. Although the divergence of ER-mediated effects in the breast, bone, and uterine endometrium has been described, a frequently overlooked site of estrogen action is the smooth muscle of the uterus. The uterine myometrium is the tissue of origin of an extremely common hormone-responsive tumor, uterine leiomyoma, a tumor with a significant impact on women's health and a possible environmental influence. This report describes an in vitro/in vivo system for identifying the effects of ER ligands in the myometrium and elucidating their mechanism of action. Several natural and synthetic xenoestrogens were evaluated at the cellular and molecular level for their ability to mimic estrogen action in uterine myometrial tissues. Diethylstilbestrol, coumestrol, genistein, naringenin, and endosulfan were able to activate the AF2 function of the ER in vitro and demonstrated agonist activity in estrogen-responsive myometrial cells, as determined by induction of proliferation and increased message levels of progesterone receptor. Compounds that could not activate AF2 function (4-hydroxy-tamoxifen, LY117018, and LY317783) did not act as estrogen agonists. For agonists, rank order of potency was predicted by receptor affinity; however, endosulfan displayed a surprising degree of activity, despite negligible receptor binding. Additionally, diethylstilbestrol and tamoxifen demonstrated prototypical agonist and antagonist effects, respectively, in the intact myometrium of sexually mature rats. The results presented here suggest that some exogenous ER ligands may mimic the effects of endogenous estrogens on uterine leiomyoma and may contribute to a complex hormonal milieu that impacts both normal and neoplastic myometrium.

Animals↗

PubMed1999

Genetic variants of the tuberous sclerosis 2 tumour suppressor gene in mouse t haplotypes.

The murine t complex on chromosome 17 contains a number of homozygous lethal and semi-lethal mutations that disrupt development of the mouse embryo. We recently characterized an embryonic lethality in the rat that results from a germ-line mutation in the tuberous sclerosis 2 (Tsc-2) tumour suppressor gene (the Eker mutation). Remarkably, mouse embryos homozygous for tw8 mutation display cranial defects reminiscent of those observed in rat embryos homozygous for the Eker mutation. To determine whether the Tsc-2 gene, which is in the t complex, is mutated in tw8 or other t haplotypes, we characterized this gene in a series of t haplotype mice. Four Tsc-2 polymorphisms were identified: three in the coding region and one intronic that appeared to be common to all t haplotypes analysed. No evidence was found to argue that the Tsc-2 gene is altered in tw8 haplotype mice. However, in the tw5 haplotype we found a G to T mutation in Tsc-2 that was present only in this t haplotype. In contrast to other polymorphisms within the Tsc-2 coding region which did not result in amino acid changes in Tsc-2 gene product tuberin, this mutation substituted a phenylalanine for a conserved cysteine in tw5 tuberin. Within the t complex, the Tsc-2 gene and the putative tw5 locus appeared to map to different positions, complicating identification of Tsc-2 as a candidate for the tw5 locus and suggesting that the G to T mutation in the Tsc-2 gene may have arisen independently of the tw5 functional mutation.

Animals↗

PubMed1999

Cyclins and cell cycle checkpoints.

The eucaryotic cell cycle is regulated by the periodic synthesis and destruction of cyclins that associate with and activate cyclin-dependent kinases. Cyclin-dependent kinase inhibitors, such as p21 and p16, also play important roles in cell cycle control by coordinating internal and external signals and impeding proliferation at several key checkpoints. Understanding how these proteins interact to regulate the cell cycle has become increasingly important to researchers and clinicians with the discovery that many of the genes that encode cell cycle regulatory activities are targets for alterations that underlie the development of cancer. Several therapeutic agents, such as DNA-damaging drugs, microtubule inhibitors, antimetabolites, and topoisomerase inhibitors, take advantage of this disruption in normal cell cycle regulation to target checkpoint controls and ultimately induce growth arrest or apoptosis of neoplastic cells. Other therapeutic drugs being developed, such as UCN-01, specifically inhibit cell cycle regulatory proteins.

Animals↗

PubMed1999

Mechanisms of epithelial development and neoplasia in the metanephric kidney.

Recent studies on the mechanisms of normal epithelial development in the kidney, and on the aetiology of renal neoplasms, are converging to reveal remarkably close relationships between the phenotypes and behaviours of normally-developing and neoplastic cells. Normal renal epithelia arise from two sources; those of the collecting duct system develop by arborisation of an initially-unbranched ureteric bud, in a manner similar to the development of other glandular organs, while epithelial nephrons develop via an unusual mesenchyme-to-epithelial transition. Both types of development require controlled proliferation, cell-cell and cell-matrix interactions, protease activity etc., but of the two tissues, the development of the nephrons is arguably the more complex. It includes many defined stages, signals and checkpoints that ensure that events happen at the right time, and that processes such as proliferation, apoptosis and differentiation are properly balanced. Detailed investigation of renal neoplasms has revealed some to be caused by mutations in molecules with known roles in normal nephrogenesis (e.g. Wilms' tumour and the WT-1 gene, renal cell carcinoma and the c-met receptor tyrosine kinase gene), some to be caused by mutations in genes expressed during normal development (e.g. renal cell carcinoma and the TSC-2 gene, renal cell carcinoma of the clear cell variety and the VHL gene). Furthermore, these and other tumours of unknown aetiology re-express genes such as Pax-2 that are expressed during the normal mesenchyme-to-epithelium transition but are shut off during terminal differentiation. Their re-appearance in tumours suggests that the cells have 'regressed' in an ontogenic sense, and their biology may therefore be understood most clearly by reference to the properties of normal developing cells rather than cells of a mature kidney.

Animals↗

PubMed1998

Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development.

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis (TSC) patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. We report that the homozygous state of this mutation (Tsc2(Ek/Ek)) was lethal in mid-gestation (the equivalent of mouse E9.5-E13.5), when Tsc2 mRNA was highly expressed in embryonic neuroepithelium. During this period homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, displayed dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupted the normal development of this tissue. Interestingly, there was significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long-Evans strain Tsc2(Ek/Ek) embryos displayed these defects whereas the Fisher 344 homozygous mutant embryos had normal-appearing neuroepithelium. Taken together, our data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain.

Animals↗

PubMed1998

Identification of a tumor-specific methylation site in the Wilms tumor suppressor gene.

Malignant mesothelioma is one of the very few extrarenal neoplasms in which the Wilms tumor suppressor gene (wt1) is expressed. We examined wt1 for alterations in rat mesotheliomas, a well characterized animal model for the human disease. Southern analysis revealed a 3.5 kb EcoRI wt1 fragment readily detectable in majority of mesothelioma cell lines and primary mesotheliomas but not in normal rat tissues. Cloning and sequencing of this fragment revealed that the presence of this EcoRI fragment resulted from an inability of this enzyme to cut at a EcoRI site in intron 1 of wt1. This site contains potential motifs for cytosine methylation and treatment of mesothelioma cells with 5-azadeoxycytosine restored the normal EcoRI digestion pattern of wt1 in these cells indicating that cleavage was inhibited by methylation at this site. Southern analysis using HpaII/MspI digestion revealed no differences in methylation between mesothelioma cell lines and normal mesothelium at other CpG sites in wt1 5' region. Renal cell carcinoma lines which did not express wt1 were also methylated at this EcoRI site. Our identification of a site frequently methylated in malignant cells, independent of gene expression, provides a new model system to study determinants of site-specific methylation in tumors.

Animals↗

PubMed1998

Magnetic resonance imaging of ultrasonic fields.

A nuclear magnetic resonance imaging (MRI) method is described that allows noninvasive, quantitative mapping of medical ultrasound (US) fields in tissue. Application of a resonant magnetic field gradient operating at the US frequency permits detection of nanometer motions associated with ultrasound, and allows direct measurement of absolute pressure, intensity, and speed of sound. By altering gradient timing, the propagation of US fields in time and space can be observed; this enables tracking of US scattering phenomena in a tissue-equivalent medium. An experimental apparatus was constructed that combined a 515-kHz focused US transducer configured with its focus in the center of a small-bore oscillating gradient. This provided an oscillating gradient with a peak gradient strength of 0.40 T/m over a useable imaging volume of 61 cm3. When used in conjunction with 1.5-T clinical MRI system, this apparatus allowed the clear visualization of the focused US field within this volume and its propagation with time. Current limits of sensitivity indicate a noise equivalent sensitivity of 3.8 nm in displacement amplitude, 19 kPa in pressure amplitude and 12 mW/cm2 in acoustic intensity. These studies indicate that MRI can provide a new, noninvasive method for US exposimetry and the basic study of ultrasound biophysics in tissue.

Agar↗

PubMed1998

Molecular cloning and characterization of a mouse gene with homology to the Duffy-antigen receptor for chemokines.

The Duffy antigen receptor for chemokines (DARC) is a receptor for both CXC and CC chemokines. We have cloned a mouse gene with a predicted amino acid sequence homology of approximately 63% to human DARC and localized this gene to mouse chromosome 1 between the Xmv41 and D1Mit166 loci. We further demonstrated that, like the human gene, the mouse gene exhibits a single intron of 462 bp which interrupts the open reading frame between the codons for the seventh and eighth amino acid residues. Northern blot analyses revealed that putative mDARC mRNA is highly expressed in adult mouse spleen and skeletal muscle and in whole embryos between embryonic days 8.5 to 12. Northern blot analysis of hemangiosarcomas which develop spontaneously in the spleen of the Eker rat reveal expression of mRNAs which hybridize with both DARC and CXCR2 probes, suggesting a potential role of these receptors in the angiogenesis associated with tumor formation.

Amino Acid Sequence↗

PubMed1997

On the preattentive accessibility of stereoscopic disparity: evidence from visual search.

We examined the generality of the claim that stereoscopic disparity is detectable in parallel across the visual field. Using a search paradigm with random-dot stereograms, we varied the relative disparity of target and distractor items. When both target and distractors had crossed disparities, both search functions (i.e., target in front of distractors and target behind distractors) were linear with positive slopes. When both target and distractors had uncrossed disparities, the pattern of results depended upon whether the target was in front of or behind the distractors-specifically, when the target was in front of the distractors, search functions were similar to those seen for "crossed" search, but when the target was behind the distractors, a nonlinear search function was found. Finally, when the target and distractors straddled the plane of fixation, a nonlinear search function was found when the target was in front of the distractors; however, when the target was behind the distractors, a linear search function with a large positive slope was found. We show that the nonlinear search functions are consistent with the effects of an intervening global surface percept. We also show that the size of the stimulus display may be a factor in some relative depth cases. Additionally, we replicate Steinman's (1987) finding that search is parallel when the distractors are located at the plane of fixation and the target disparity is crossed, eliminating monocular and spatial cues to target presence that may have been present in his original study. In a final control experiment, we showed that reaction times did not increase with set size when observers performed another kind of perceptual task on similar random-dot stereogram displays. This eliminates the possibility that some of the results obtained here can be explained by increases in the difficulty of perceiving/fusing the stimuli when the number of distractors is increased.

Adult↗

PubMed1997

Characterization of the rat neurofibromatosis 2 gene and its involvement in asbestos-induced mesothelioma.

The neurofibromatosis 2 (NF2) tumor suppressor gene was recently implicated in the genesis of human mesothelioma. To investigate the role of this tumor suppressor gene in rat asbestos-induced mesothelioma, a commonly used model for the human disease, we characterized the rat homologue of NF2 and examined rat chrysotile-induced primary mesotheliomas and cell lines derived from chrysotile- and crocidolite-induced mesotheliomas for alterations in this gene. The coding sequence obtained for the rat NF2 gene had 90% nucleotide homology with the human NF2 gene. The rat NF2 gene was ubiquitously expressed as a 4.4-kb transcript in normal rat tissues as well as in rat mesothelioma cell lines. Reverse transcription-polymerase chain reaction analysis to examine splicing of NF2 exons in mesothelioma cells indicated that the exon splicing pattern was similar in normal and neoplastic cells. To determine if mutations had occurred in the NF2 coding region in rat mesotheliomas, single-strand conformation polymorphism analysis and direct sequencing were used to screen 10 primary tumors and six tumor cell lines. No DNA sequence alterations were observed in any of the rat mesothelioma samples examined. These findings contrast with data reported previously for human mesotheliomas, in which the NF2 gene was found to be mutated in 40% of cases. Taken together, these data suggest that the role of NF2 in the development of rodent asbestos-induced mesothelioma may differ significantly from the role in the human disease.

Animals↗

PubMed1997

Altered expression of transforming growth factor-alpha: an early event in renal cell carcinoma development.

Transforming growth factor-alpha (TGF-alpha) is a multifunctional cell regulatory protein with a wide range of effects on cell growth and differentiation and has been implicated in the neoplastic transformation of a variety of cell types. Altered expression of TGF-alpha and its cognate receptor (epidermal growth factor receptor) is enhanced in human and rat renal cell carcinomas. The objective of the study reported here was to determine whether altered TGF-alpha expression is an early or late event in renal tubular oncogenesis. The immunohistochemical expression of TGF-alpha was studied in preneoplastic renal tubular lesions in a rat model of hereditary renal cell carcinoma. Strong TGF-alpha immunoreactivity was present at all stages of renal cell tumor development, including the earliest detectable dysplasias. In contrast, the non-neoplastic regenerating tubular epithelium of rat degenerative nephropathy did not stain for TGF-alpha, although this tissue exhibited a proliferative capacity similar to that observed in the dysplastic and neoplastic lesions. This study indicated that altered TGF-alpha expression was detectable early in the development of renal cell tumors and may be an important feature of the transformed phenotype.

Animals↗

PubMed1996

Inhibition of hamster mesothelioma tumorigenesis by an antisense expression plasmid to the insulin-like growth factor-1 receptor.

We evaluated the effect of antisense insulin-like growth factor (IGF) receptor transcripts on the proliferation and tumorigenicity in an SV40-induced, immunocompetent hamster mesothelioma model (H9A). Expression of IGF-1 and IGF-1 receptor (IGF-1R) genes was identified from H9A RNA using reverse transcription-PCR and Northern analysis. H9A cells were electroporated with inducible expression vectors (under the transcriptional control of heat shock promoter HSP70) containing a cDNA fragment corresponding to base pairs 1-309 of IGF-1R in the sense or antisense orientation to generate the respective clones A3 sense or B9 antisense. The expression vector in genomic DNA was detected with PCR analysis as a 173-bp fragment on ethidium bromide gels. The effects of the expression vectors were then evaluated in vitro under active (at 39 degrees C) or inactive (at 34 degrees C) conditions. At 39 degrees C, the B9 antisense transfectants demonstrated significantly less proliferation than A3 sense transfectants (P2 < 0.02). At 34 degrees C, cell growth of A3 sense- and B9 antisense-transfected cells was not significantly different. In vivo tumorigenicity was evaluated in hamsters inoculated with 10(5) A3 sense- or B9 antisense-transfected cells. The A3 sense clones resulted in greater numbers of tumors in vivo compared to the B9 antisense clone (P2 = 0.0001). When genomic DNA from tumors that developed in A3 sense and B9 antisense animals was analyzed for the expression vectors, a 173-bp fragment amplified from the expression vector was identified in the sense tumors but not in antisense B9 or wild-type H9A tumors, indicating a loss of the vector from the antisense clones that proliferated in vivo. The inhibitory effect of IGF-1R antisense transcripts on hamster mesothelioma demonstrated in this study by decreased growth and tumorigenicity in vitro and in vivo may have implications for the therapy of human mesothelioma.

Animals↗

PubMed1996

Cancer susceptibility genes and molecular carcinogenesis. American Association for Cancer Research Special Conference: Cancer Susceptibility Genes and Molecular Carcinogenesis.

As elaborated by Nobel Laureate Harold Varmus (Director of the National Institutes of Health, Bethesda, MD, USA) in his Keynote address, future studies must not only continue to expand our repertoire of genetic determinants of cancer susceptibility by identifying new cancer susceptibility genes, but most also now begin to address the function of these genes and their roles in the process of tumor development. Key areas for future investigation in this field, identified at the meeting, were: (1) understanding how susceptibility genes are altered in cancer cell physiology, (2) elucidating the function of these genes in the biochemistry of cellular signaling pathways; and (3) improving our ability to use this information to extrapolate basic oncology research to the clinic. At this meeting, the fields of molecular carcinogenesis, DNA repair and human genetics were very successfully integrated. These fields have converged in the areas of cell cycle control and genetic susceptibility, and this meeting capitalized on this convergence to highlight recent progress in these areas. Key areas for future research were also emphasized, including the need to apply the rapidly emerging information from these fields towards developing novel therapeutic modalities to treat and prevent cancer.

Aging↗

PubMed1995

Expression of insulin-like growth factor II in spontaneously immortalized rat mesothelial and spontaneous mesothelioma cells: a potential autocrine role of insulin-like growth factor II.

Insulin-like growth factors (IGFs) are polypeptides that play an important role in cellular proliferation and differentiation. The present study examines the role of IGFs in the growth of mesothelial cells. Cell lines derived from normal rat mesothelium as well as lines derived from spontaneous rat mesotheliomas were found to express RNA transcripts for IGF-II. In contrast, cell lines derived from asbestos-induced rat mesotheliomas did not express this growth factor. All cell lines expressed receptors for IGF-I and IGF-II, as well as insulin receptors. Coexpression of IGF-II and its cognate receptor suggested that IGF-II was acting as an autocrine growth factor in the spontaneously immortalized cells and the cells derived from the spontaneous tumors. The biological activity of IGF-II secreted by the cell lines into conditioned medium could be neutralized using an IGF-II-specific antibody. Growth was inhibited in a dose-dependent manner; at the highest antibody concentration used (100 micrograms anti-IGF-II/ml), cell growth was decreased to 47% of control values. This inhibition was partially reversible by treatment of the cultures with IGF-II (91% of the control). These data suggest that IGF-II expression may be involved in the spontaneous alteration of rat mesothelial cells and may function as an autocrine or paracrine growth factor to modulate the growth of these cells in vitro and in vivo. Ubiquitous expression of IGF-II by cells that have not been exposed to asbestos and the lack of IGF-II expression by asbestos-transformed cells suggest that the mechanisms of changes in growth factor expression differ in mesothelial cells transformed by different pathways.

Animals↗

PubMed1995

Association of rat p15INK4B/p16INK4 deletions with monosomy 5 in kidney epithelial cell lines but not primary renal tumors.

Recently the putative tumor suppressor gene p16INK4 was mapped to human chromosome 9p21, which is homologous to rat chromosome 5. Monosomy of rat chromosome 5 occurs with high frequency in rat kidney tumor-derived cell lines (ERC lines). Thus, we studied these lines in order to investigate the involvement of p15INK4B and p16INK4 in the genesis of this tumor type. p15INK4B and p16INK4 were found by Southern blot analysis to be codeleted in five of seven of these lines. This was confirmed by Northern blot analysis with a probe for the rat p15INK4B gene. In normal rat tissues, expression of p15INK4B was abundant in lung (2.5 and 2.0 kilobases), less abundant in testis (2.5, 2.0, 1.1, and 0.9 kilobases), barely detectable in liver (2.0 kilobases), and not detectable in neonatal kidney, adult kidney, brain, heart, or spleen. In the ERC lines, p15INK4B was expressed as a single 2.0-kilobase transcript observed only in those cell lines in which the gene was detected by Southern blot analysis. However, neither p15INK4B nor p16INK4 were deleted in 12 of 12 primary kidney tumors examined, suggesting that deletion of these genes is not directly involved in the process of renal tumor development but may be related to tumor progression or autonomous growth in vitro. A panel of rat kidney epithelial cell lines chemically transformed in vitro (TRKE lines) that had high-frequency monosomy 5 were also examined, but deletion of p15INK4B and p16INK4 was observed in only one of six of the TRKE lines. To our knowledge, this is the first reported investigation of these genes in rodent tumors and cell lines, and its data support the theory that alterations of genes located in the INF region of rat chromosome 5 may play a role in rodent cell transformation.

Animals↗

PubMed1995

Allelic loss at the tuberous sclerosis 2 locus in spontaneous tumors in the Eker rat.

Somatic events leading to the inactivation of tumor suppressor genes often involve chromosomal alterations that can be detected as loss of heterozygosity (LOH). In the Eker rat, spontaneous tumors of the kidney, uterus, and spleen develop as a result of a germline mutation of the tuberous sclerosis 2 (Tsc2) gene. We examined the pattern and frequency of LOH at the predisposing locus in 77 primary tumors and cell lines to gain an understanding of the role of Tsc2 allelic loss in the pathogenesis of Eker-derived tumors. Although most renal and uterine tumors (primary and cell lines) displayed LOH, splenic hemangiosarcomas did not. Although the presence of normal tissue may account for some of this difference, the possibility exists that an alternative mechanism, such as subtle mutation or gene dosage effects, may be involved during splenic tumorigenesis. Northern analysis confirmed that LOH resulted in loss of the wild-type transcripts for the Tsc2 gene. Thus, the inactivation of both alleles plays an important role in renal and uterine tumor development, in keeping with Knudson's two-hit hypothesis. In addition, renal tumors that retained the wild-type allele also did not express the normal transcript, suggesting that the remaining Tsc2 alleles had acquired subtle mutations resulting in loss of gene function.

Alleles↗

PubMed1995

Hereditary renal cell carcinoma in the Eker rat: a unique animal model for the study of cancer susceptibility.

A class of genes, the so-called tumor suppressor genes or anti-oncogenes, was originally identified as being responsible for germ-line transmission of cancer susceptibility in humans. Tumor suppressor genes are recessive at the cellular level with respect to oncogenesis but often manifest as dominantly inherited familial cancer syndromes. This type of cancer syndrome arises in the Eker rat due to a genetic defect in the tuberous sclerosis 2 (Tsc2) gene. The Eker rat familial cancer syndrome serves as a unique animal model in which to study the molecular pathways of renal tubular epithelial carcinogenesis as well as a valuable tool for studies that examine how chemical carcinogens interact with cancer susceptibility genes.

Animals↗