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J C Barrett

Publications and source records attributed to J C Barrett.

At least 73 records · Page 4Linked to original sources
PubMed2000

Complementation of an hMSH2 defect in human colorectal carcinoma cells by human chromosome 2 transfer.

The human colorectal tumor cell line LoVo has a homozygous deletion in the hMSH2 gene from exon 3 to exon 8, is deficient in mismatch repair (MMR) activity, and exhibits microsatellite instability. To determine whether the introduction of a wild type hMSH2 into LoVo restores MMR activity and stabilizes microsatellite loci, we transferred a chromosome 2 fragment containing hMSH2 into a homologous recombination-proficient chicken DT40/human hybrid (DT40 2C) and a human chromosome 2 in a mouse A9 hybrid to LoVo. Transfers of these chromosomes into LoVo resulted in LoVo both with and without a wild-type hMSH2. Complete correlation was found between the presence of the wild-type hMSH2 and hMSH2 expression, an increased stability in microsatellite loci, and competency in MMR. The hMSH2-positive LoVo hybrids also showed an increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine. This enhanced toxicity is associated with G(2) cell-cycle arrest followed by premature mitosis and cell death. These results suggest that hMSH2 may be responsible for complementing mutator and drug-resistant phenotypes in chromosome 2-transferred LoVo cells. To test whether the hMSH2 in DT40 2C cells can be modified by homologous recombination, we transfected DT40 2C with a targeting vector containing an hMSH2 exon 4 disrupted by the zeocin-resistant gene. The results showed that the hMSH2 locus in DT40 2C was efficiently targeted by an exogeneously transfected homologous sequence, suggesting that transfer of a modified hMSH2 from DT40 2C to LoVo via chromosome transfer could be used to determine the function of hMSH2.

Animals↗
PubMed2000

Mapping of metastasis suppressor genes for prostate cancer by microcell-mediated chromosome transfer.

AIM: To identify the metastasis suppressor genes for prostate cancer. METHODS: A copy of human chromosomes was introduced into the highly metastatic Dunning R-3327 rat prostate cancer cells by the use of microcell-mediated chromosome transfer. Relationships between the size of human chromosomes introduced into microcell hybrid clones and the number of lung metastases produced by the clones were analyzed to determine which part of human chromosomes contained the metastasis suppressor gene(s) for prostate cancer. To determine portions of human chromosomes introduced, G-banding chromosomal analysis, fluorescence in situ hybridization analysis, and polymerase chain reaction analysis were performed. RESULTS: Each of microcell hybrid clones containing human chromosomes 7, 8, 10, 11, 12, or 17 showed decreased ability to metastasize to the lung without any loss of tumorigenicity. This demonstrates that these human chromosomes contain metastasis suppressor genes for prostate cancer. Spontaneous deletion of portions of human chromosomes was observed in the human chromosome 7, 10, 11, 12, and 17 studies. In the human chromosome 8 study, irradiated microcell-mediated chromosome transfer was performed to enrich chromosomal arm deletions of human chromosome 8. Molecular and cytogenetic analyses of microcell hybrid clones demonstrated that metastasis suppressor genes on human chromosomes were located on 7q21-22, 7q31.2-32, 8p21-12, 10q11-22, 11p13-11.2, 12p11-q13, 12q24-ter, and 17pter-q23. KAI1 and MKK4/SEKI were identified as metastasis suppressor genes from 11p11.2 and 17p12, respectively. CONCLUSION: This assay system is useful to identify metastasis suppressor gene (s) for prostate cancer.

Animals↗
PubMed2000

N-acetyl-L-cysteine simultaneously increases mitogenesis and suppresses apoptosis in mitogen-stimulated B-lymphocytes from p53 haploinsufficient Tg.AC (v-Ha-ras) mice.

Recent epidemiological evidence suggests that antioxidants may enhance carcinogenesis by promoting cellular proliferation and/or impeding programmed cell death. We examined the effect of N-acetyl-l-cysteine (NAC) on mitogenesis and apoptosis in splenocytes from p53 haploinsufficient Tg.AC (v-Ha-ras) mice. This model contains genetic lesions found frequently in human cancer and is predisposed to develop carcinogen-induced cancer. Splenocytes were incubated with NAC alone or with the B- and T-cell-specific mitogens Concanavalin A (Con A) and E. coli lipopolysaccharide (LPS), respectively. Mitogenesis increased 17-fold in mitogen-stimulated cultures and 10-fold in cultures incubated with NAC alone. Co-incubation with both NAC (1000 microg/mL) and mitogen increased mitogenesis by 33-fold without changing apoptosis rates. Strikingly, incubation with NAC and LPS attenuated LPS-induced apoptosis. Mitogen alone did not affect GSH levels but NAC-induced increases were significantly depleted by co-incubation with mitogen. Furthermore, NAC increased the number of CD45R+ B cells, but decreased CD3+ T cells showing enhanced survival of B cells under these conditions. These results demonstrate concurrent reduced apoptosis and increased mitogenesis in B lymphocytes that may favor clonal selection of preneoplastic cells.

Acetylcysteine↗
PubMed1999

DNA methylation analysis of the promoter region of the human telomerase reverse transcriptase (hTERT) gene.

The promoter of the hTERT gene encoding the catalytic subunit of telomerase was recently cloned and has a dense CG-rich CpG island, suggesting a role for methylation in regulation of hTERT expression. In this study, we have initiated the analysis of the regulation of hTERT expression by examining the methylation status of up to 72 CpG sites extending from 500 bases upstream of the transcriptional start site of the hTERT gene into the first exon in 37 cell lines. These cell lines represent a variety of cell and tissue types, including normal, immortalized, and cancer cell lines from lung, breast, and other tissues. Using bisulfite genomic sequencing, we did not find a generalized pattern of site-specific or region-specific methylation that correlated with expression of the hTERT gene: most of the hTERT-negative normal cells and about one-third of the hTERT-expressing cell lines had the unmethylated/hypomethylated promoter, whereas the other hTERT-expressing cell lines showed partial or total methylation of the promoter. The promoter of one hTERT-negative fibroblast cell line, SUSM-1, was methylated at all sites examined. Treatment of SUSM-1 cells with the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A induced the cells to express hTERT, suggesting a potential role for DNA methylation and/or histone deacetylation in negative regulation of hTERT. This study indicates that there are multiple levels of regulation of hTERT expression in CpG island methylation-dependent and -independent manners.

Cell Line↗
PubMed1999

Loss of KAI1 expression in the progression of colorectal cancer.

The transmembrane 4 superfamily member KAI1 (CD82) has been shown to inhibit pulmonary metastases in experimental metastasis models of prostate cancer and melanoma. KAI1 expression is decreased in the progression of common solid epithelial tumors of adulthood, including lung, prostate, breast, esophageal, gastric, pancreatic, and bladder cancers. The purpose of our study was to investigate KAI1 expression in the progression of human colorectal cancer. We first analyzed 20 colorectal cancer cell lines by immunoblot techniques. KAI1 was expressed heterogeneously, with the tumor cell lines having a more complex degree of glycosylation compared with that of the normal colonic tissue. KAI1 was highly expressed in the primary SW480 colon cancer cell line but was down-regulated 15-fold in the matched metastatic SW620 cell line. We also investigated KAI1 protein expression by immunohistochemistry in tissues from 84 patients with colorectal cancer. Each tissue section was assigned a KAI1 mean score (KMS) from 0 to 300 based on the product of the percentage of cells that stained for KAI1 and the intensity of the stain (1, 2, or 3). In 84 patients with colorectal cancer, KAI1 was expressed at high levels in normal colonic mucosa (KMS 226) but was expressed at lower levels in the primary tumors (KMS 65; P < 0.0001). In a subset of 12 patients with stage IV metastatic disease, we observed a progressive down-regulation of KAI1, from the normal adjacent colonic mucosa (KMS 193) to the primary tumor (KMS 72; P = 0.0001) to the liver metastasis (KMS 25; tumor compared with metastasis, P = 0.0135). We found no correlation between loss of KAI1 expression and stage of disease. In 10 patients, we also noted loss of KAI1 expression in the transition from normal colonic mucosa (KMS 237) to adenoma (KMS 174) to carcinoma (KMS 62; P < 0.0167 for all three comparisons). We conclude that the down-regulation of KAI1 occurs early in the progression of colorectal cancer.

Adenoma↗
PubMed1999

Application of complementary DNA microarray technology to carcinogen identification, toxicology, and drug safety evaluation.

One major challenge facing today's cancer researchers and toxicologists is the development of new approaches for the identification of carcinogens and other environmental hazards. Here, we describe the potential impact of emerging technologies for measuring gene expression profiles on carcinogen identification and on the general field of toxicology. An example of one of these technologies is the use of cDNA microarray chips. We provide an overview to the key questions that are confronting investigators charged with determining the relative safety of natural or synthetic chemicals to which humans are exposed, followed by a discussion of how cDNA microarray technology may be applied to these questions. Gene chip technology is still a relatively new technology, and only a handful of studies have demonstrated its utility. However, as the technical hurdles to development are passed, the use of this methodology in addressing the questions raised here will be critical to increase the sensitivity of detection of the potential toxic effects of environmental chemicals and to understand their risks to humans.

Carcinogens↗
PubMed1999

Mutation of beta-catenin is an early event in chemically induced mouse hepatocellular carcinogenesis.

beta-catenin activation, and subsequent upregulation of Wnt-signaling, is an important event in the development of certain human and rodent cancers. Recently, mutations in the beta-catenin gene in the region of the serine-threonine glycogen kinase (GSK)-3beta phosphorylation target sites have been identified in hepatocellular neoplasms from humans and transgenic mice. In this study we examined 152 hepatocellular neoplasms from B6C3F1 mice included in five chemical treatment groups and controls for mutations in the beta-catenin gene. Twenty of 29 hepatocellular neoplasms from mice treated with methyleugenol had point mutations at codons 32, 33, 34 or 41, sites which are mutated in colon and other cancers. Likewise, nine of 24 methylene chloride-induced hepatocellular neoplasms and 18 of 42 oxazepam-induced neoplasms exhibited similar mutations. In contrast, only three of 18 vinyl carbamate-induced liver tumors, one of 18 TCDD-induced liver tumors, and two of 22 spontaneous liver neoplasms had mutations in beta-catenin. Thus, there appears to be a chemical specific involvement of beta-catenin activation in mouse hepatocellular carcinogenesis. Expression analyses using Western blot and immunohistochemistry indicate that beta-catenin protein accumulates along cell membranes following mutation. The finding of mutations in both adenomas and carcinomas from diverse chemical treatment groups and the immunostaining of beta-catenin protein in an altered hepatocellular focus suggest that these alterations are early events in mouse hepatocellular carcinogenesis.

Adenoma, Liver Cell↗
PubMed1999

Reduced capacitative calcium entry correlates with vesicle accumulation and apoptosis.

A preneoplastic variant of Syrian hamster embryo cells, sup(+), exhibits decreased endoplasmic reticulum calcium levels and subsequently undergoes apoptosis in low serum conditions (Preston, G. A., Barrett, J. C., Biermann, J. A., and Murphy, E. (1997) Cancer Res. 57, 537-542). This decrease in endoplasmic reticulum calcium appears to be due, at least in part, to reduced capacitative calcium entry at the plasma membrane. Thus we investigated whether inhibition of capacitative calcium entry per se could reduce endoplasmic reticulum calcium and induce apoptosis of cells. We find that treatment with either SKF96365 (30-100 microM) or cell-impermeant 1,2-bis(o-amino-5-bromophenoxy)ethane-N,N,N', N'-tetraacetic acid (5-10 mM) is able to induce apoptosis of cells in conditions where apoptosis does not normally occur. Because previous work has implicated vesicular trafficking as a mechanism of regulating capacitative calcium entry, we investigated whether disruption of vesicular trafficking could lead to decreased capacitative calcium entry and subsequent apoptosis of cells. Coincident with low serum-induced apoptosis, we observed an accumulation of vesicles within the cell, suggesting deregulated vesicle trafficking. Treatment of cells with bafilomycin (30-100 nM), an inhibitor of the endosomal proton ATPase, produced an accumulation of vesicles, decreased capacitative entry, and induced apoptosis. These data suggest that deregulation of vesicular transport results in reduced capacitative calcium entry which in turn results in apoptosis.

Animals↗
PubMed1999

Two posttranscriptional pathways that regulate p21(Cip1/Waf1/Sdi1) are identified by HPV16-E6 interaction and correlate with life span and cellular senescence.

The p21((Cip1/Waf1/Sdi1)) protein is a cyclin-dependent kinase inhibitor that is induced in normal human fibroblasts (NHF) following DNA damage, following serum stimulation, and at cellular senescence. Expression of the human papilloma virus 16 E6 oncoprotein in NHF cells results in the loss of the p21 protein, independent of mRNA level under most conditions. The p21 protein levels in NHF-E6 cells remained low following DNA damage or serum stimulation even though mRNA levels increased. In contrast, the p21 protein was transiently induced in NHF-E6 cells at the onset of cellular senescence. Expression of the E6 oncoprotein in transformed cells had no effect on p21 protein levels. This demonstrates that two posttranscriptional pathways regulate expression of p21 protein in NHF cells under different conditions. Disruption of posttranscriptional regulation is correlated with extension of life span, altered cell fate, and transformation.

Cell Transformation, Viral↗
PubMed1999

Cloning and characterization of the promoter region of human telomerase reverse transcriptase gene.

Activation of telomerase is one of the rate-limiting steps in human cell immortalization and carcinogenesis Human telomerase is composed of at least two protein subunits and an RNA component. Regulation of expression of the catalytic subunit, human telomerase reverse transcriptase (hTERT), is suggested as the major determinant of the enzymatic activity. We report here the cloning and characterization of the 5'-regulatory region of the hTERT gene. The highly GC-rich content of the 5' end of the hTERT cDNA spans to the 5'-flanking region and intron 1, making a CpG island. A 1.7-kb DNA fragment encompassing the hTERT gene promoter was placed upstream of the luciferase reporter gene and transiently transfected into human cell lines of fibroblastic and epithelial origins that differed in their expression of the endogenous hTERT gene. Endogenous hTERT-expressing cells, but not nonexpressing cells, showed high levels of luciferase activity, suggesting that the regulation of hTERT gene expression occurs mainly at the transcriptional level. Additional luciferase assays using a series of constructs containing unidirectionally deleted fragments revealed that a 59-bp region (-208 to -150) is required for the maximal promoter activity. The region contains a potential Myc oncoprotein binding site (E-box), and cotransfection of a c-myc expression plasmid markedly enhanced the promoter activity, suggesting a role of the Myc protein in telomerase activation. Identification of the regulatory regions of the hTERT promoter sequence will be essential in understanding the molecular mechanisms of positive and negative regulation of telomerase.

Base Sequence↗
PubMed1999

Suppression of the tumorigenicity of prostatic cancer cells by gene(s) located on human chromosome 19p13.1-13.2.

BACKGROUND: In previous reports, we used microcell fusion-mediated chromosomal transfer to introduce normal human chromosomes into highly metastatic rat prostatic cancer cells to map the location of tumor and metastasis suppressor genes. The gene for prostate-specific antigen as well as several classes of genes, including cell adhesion molecules, previously demonstrated to be altered during prostate cancer progression, were mapped to human chromosome 19. METHODS: A normal human chromosome 19 was introduced into Dunning-R3327 AT6.1 rat and TSU-prl human prostatic cancer cells by microcell-mediated chromosome transfer to test the suppressive effects of this chromosome on prostate cancer. Five independent hybrid clones from Dunning-R3327 AT6.1 rat prostatic cancer cells and four independent hybrid clones from TSU-pr1 human prostatic cancer cells were isolated, karyotyped, allelotyped, and analyzed for in vitro and in vivo growth characteristics. RESULTS: Introduction of human chromosome 19 into both the rat and human prostatic cancer cells resulted in alteration of cell morphology in vitro and suppression of tumorigenicity in vivo in athymic nude mice. Highly polymorphic SSR2 markers mapped to human chromosome 19 were used to determine the portions of human chromosome 19 retained in the hybrids. These analyses identified a region localized on human chromosome 19p13.1-13.2 that is responsible for the tumor suppression of both rat and human prostatic cancer cells. The expression of several genes previously mapped to this human chromosome 19p13.1-13.2 region (i.e., ICAM-1, Notch3, and Stau) were analyzed to evaluate if they could be candidate suppressor genes for prostate cancer cell growth in vivo, but no expression patterns consistent with those predicted for a suppressor gene were observed. CONCLUSIONS: Human chromosome 19p13.1-13.2 contains potential tumor suppressor gene(s) for prostate cancer.

Animals↗
PubMed1999

Metastasis suppressor gene(s) for rat prostate cancer on the long arm of human chromosome 7.

Allelotype analyses of human prostate cancer indicate that allelic losses on human chromosome arms 7q, 8p, 10q, 13q, 16q, 17q, and 18q are observed frequently. For the study of the possible biological significance of the frequently observed deletions on chromosome arm 7q in human prostate cancer, human chromosome 7 was introduced into highly metastatic rat prostate cancer cells by use of a microcell-mediated chromosome transfer technique. The introduction of human chromosome 7 resulted in the suppression of metastatic ability of the microcell hybrids, whereas no suppression of tumorigenicity was observed. To identify the portion of chromosome 7 containing the metastasis-suppressive function gene, the derivative chromosome 7 that was generated with the initial transfer was retransferred into rat prostate cancer cells. Human chromosome 7-containing rat prostate cancer cells could be used as the donor cells, because rodent cells produced a sufficient number of microcells with colchicine treatment. Cytogenetic and molecular analyses of these clones demonstrated that loss of segments on 7q was related to the reexpression of the metastatic phenotype. These results show that human 7q contains a metastasis suppressor gene or genes for rat prostate cancer. The findings also suggest that this gene may play an important role in the progression of human prostate cancer.

Animals↗
PubMed1999

Interference of proteins involved in the cytoplasmic sequestration of p53 with human papillomavirus E6-mediated degradation.

The oncogenic human papillomaviruses (HPVs) are able to efficiently target p53 for degradation by the ubiquitin pathway. We previously demonstrated inefficient HPV E6-mediated degradation and resulting high steady-state levels of p53 in cell hybrids between a peripheral neuroepithelioma cell line and a cervical carcinoma cell line (HeLa). We now show that the p53 protein in these cell hybrids was cytoplasmically sequestered and exhibited sporadic punctate staining, which is characteristic of the p53 expression pattern observed in neuroblastic neuroblastoma (NB) cell lines, in which p53 is also sequestered. We hypothesized that the cytoplasmic sequestration of p53 in the cell hybrids might correlate with its inability to be rapidly degraded by HPV E6. Using NB cell lines as a model system to test this hypothesis, we demonstrated that the introduction of HPV E6 into two NB cell lines resulted in p53 insensitivity to HPV E6-mediated degradation. This was assessed by both pulse-chase analysis of p53 in metabolically labeled NB cells and western blotting. The enhanced stability of p53 was not due to a lack of HPV E6 expression or to a mutant conformation of the p53 protein. Our results therefore suggest that proteins involved in the cytoplasmic sequestration of p53 may also interfere with the ability of HPV E6 to target p53 for degradation.

Breast Neoplasms↗
PubMed1999

Microarrays and toxicology: the advent of toxicogenomics.

The availability of genome-scale DNA sequence information and reagents has radically altered life-science research. This revolution has led to the development of a new scientific subdiscipline derived from a combination of the fields of toxicology and genomics. This subdiscipline, termed toxicogenomics, is concerned with the identification of potential human and environmental toxicants, and their putative mechanisms of action, through the use of genomics resources. One such resource is DNA microarrays or "chips," which allow the monitoring of the expression levels of thousands of genes simultaneously. Here we propose a general method by which gene expression, as measured by cDNA microarrays, can be used as a highly sensitive and informative marker for toxicity. Our purpose is to acquaint the reader with the development and current state of microarray technology and to present our view of the usefulness of microarrays to the field of toxicology.

Animal Testing Alternatives↗
PubMed1999

Involvement of mutations in the DPC4 promoter in endometrial carcinoma development.

To define the target of chromosome 18q loss of heterozygosity, which is prevalent in endometrial carcinomas, we made a deletion map from 64 tumors. Loss of heterozygosity on 18q was found in 20 tumors. Among these, 14 tumors carried deletions at the 18q21.1 region, where the DPC4 gene is located. DPC4 transcription was disturbed in all six of the tumors with deletions at 18q21.1 examined, which sharply contrasted with the positive transcription in 12 tumors that retained heterozygosity at the 18q21.1 region. However, in the 14 tumors with the 18q21.1 deletions, the remaining allele had the wild-type sequence of the DPC4 coding region instead of somatic mutations in the DPC4 coding region. We found a one- and two-base substitutions in the DPC4 promoter in two of the six tumors that showed disturbed DPC4 transcription. Chloramphenicol acetyltransferase assays clearly demonstrated that the mutant promoters had the potential to suppress or silence DPC4 transcription, implicating the DPC4 gene in endometrial carcinoma.

Alleles↗
PubMed1999

Telomerase-independent senescence of human immortal cells induced by microcell-mediated chromosome transfer.

Maintenance of telomeres, commonly through expression of telomerase activity, is necessary but may not be sufficient for human cells to escape from the cellular senescence program and become immortal. We report here that human tumor cells could undergo cellular senescence in the presence of telomerase activity when a specific normal human chromosome was introduced via microcell-mediated chromosome transfer. The cell models studied include SiHa (uterine cervical carcinoma cells expressing E6 and E7 oncoproteins of human papillomavirus type 16) with a transferred chromosome 2, CC1 (choriocarcinoma cells expressing an amino-terminally truncated p53 protein) with a transferred chromosome 7, and JTC-32 (bladder carcinoma cells) with a transferred chromosome 11. The microcell hybrids with the indicated chromosomes ceased to divide after five to 10 population doublings and showed senescence-associated beta-galactosidase activity but still expressed the genes encoding three components of human telomerase, consistent with the retention of telomerase activity. These results are evidence for barriers to human cell immortalization, which involve activation of unidentified senescence-inducing genes that function independently of inactivation of telomerase.

Cell Line, Transformed↗