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Biomedical subjects

C R Boland

Publications and source records attributed to C R Boland.

At least 55 records · Page 3Linked to original sources
PubMed1997

Localization of the Bannayan-Riley-Ruvalcaba syndrome gene to chromosome 10q23.

BACKGROUND & AIMS: Bannayan-Riley-Ruvalcaba syndrome is a congenital syndrome with characteristic features of macrocephaly, cognitive and motor dysfunction, subcutaneous and visceral lipomas and hemangiomas, and intestinal juvenile polyposis. It has been suggested that Bannayan-Riley-Ruvalcaba syndrome may be a variant of juvenile polyposis coli because of the shared features of intestinal juvenile polyps. The aim of this study was to precisely map loss of DNA from 2 patients with intestinal juvenile polyposis and karyotypic abnormalities involving chromosome 10q. METHODS: DNA was extracted from peripheral leukocytes drawn from each patient and each patient's biological parents. The DNA was amplified by polymerase chain reaction using primers specific for microsatellites located on chromosome 10q. RESULTS: Precise mapping localized a maximal distance of 1.0 cM that was commonly deleted from each patient's genome, between D10S541 and D10S1735. This area overlaps the region for Cowden disease, a distinct hamartomatous intestinal polyposis syndrome with increased risk of breast and thyroid carcinoma. CONCLUSIONS: The three hamartomatous polyposis syndromes, Bannayan-Riley-Ruvalcaba syndrome, juvenile polyposis coli, and Cowden disease, may share the same genetic defect because of their common map localization to chromosome 10q23.

Adenomatous Polyposis Coli↗
PubMed1996

Expression of human MutS homolog 2 (hMSH2) protein in resting and proliferating cells.

The hMSH2 protein plays an important role in the DNA mismatch repair system. Since this system is involved in the correction of errors that occur during DNA replication, we studied the expression of hMSH2 protein in resting and DNA-replicating cells, as well as through the cell cycle in cell types with different growth characteristics. Using Western blot analysis, we showed that hMSH2 protein was detectable in resting peripheral blood lymphocytes and thymocytes. However, when these cells were induced to proliferate, the protein level increased at least 12-fold. In cell-cycle dependent expression studies we chose two DNA mismatch repair proficient cell lines (HEL and HeLa-S3), and flow cytometry was used to monitor cell-cycle progression. At every phase in the cell cycle, the steady-state level of hMSH2 was higher than in resting lymphocytes or thymocytes, and only minor variations of expression level were observed through the cell cycle. In particular, a two to fourfold decrease in hMSH2 expression occurred at G1 in HEL and at early S phase in HeLa-S3, but higher expression levels resumed during the replicative and postreplicative phases of the cell cycle. Interestingly, hMSH2 protein expression decreased fourfold when HEL cells were induced to differentiate along the megakaryocyte lineage, when continuous DNA replication occurs without mitosis. These results suggest that a basal level of hMSH2 protein expression is necessary for resting and differentiated cells, and that increased hMSH2 protein expression is required when DNA replication is activated and followed by mitosis.

Cell Cycle↗
PubMed1996

Limited up-regulation of DNA methyltransferase in human colon cancer reflecting increased cell proliferation.

Epigenetic alterations in the genome of tumor cells have attracted considerable attention since the discovery of widespread alterations in DNA methylation of colorectal cancers over 10 years ago. However, the mechanism of these changes has remained obscure. el-Deiry and coworkers [el-Deiry, W. S., Nelkin, B. D., Celano, P., Yen, R. C., Falco, J. P., Hamilton, S. R. & Baylin, S. B. (1991) Proc. Natl. Acad. Sci. USA 88, 3470-3474], using a quantitative reverse transcription-PCR assay, reported 15-fold increased expression of DNA methyltransferase (MTase) in colon cancer, compared with matched normal colon mucosa, and a 200-fold increase in MTase mRNA levels compared with mucosa of unaffected patients. These authors suggested that increases in MTase mRNA levels play a direct pathogenetic role in colon carcinogenesis. To test this hypothesis, we developed a sensitive quantitative RNase protection assay of MTase, linear over three orders of magnitude. Using this assay on 12 colorectal carcinomas and matched normal mucosal specimens, we observed a 1.8- to 2.5-fold increase in MTase mRNA levels in colon carcinoma compared with levels in normal mucosa from the same patients. There was no significant difference between the normal mucosa of affected and unaffected patients. Furthermore, when the assay was normalized to histone H4 expression, a measure of S-phase-specific expression, the moderate increase in tumor MTase mRNA levels was no longer observed. These data are in contrast to the previously reported results, and they indicate that changes in MTase mRNA levels in colon cancer are nonspecific and compatible with other markers of cell proliferation.

Colon↗
PubMed1996

Competency in mismatch repair prohibits clonal expansion of cancer cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

The phenomenon of alkylation tolerance has been observed in cells that are deficient in some component of the DNA mismatch repair (MMR) system. An alkylation-induced cell cycle arrest had been reported previously in one MMR-proficient cell line, whereas a MMR-defective clone derived from this line escapes from this arrest. We examined human cancer cell lines to determine if the cell cycle arrest were dependent upon the MMR system. Growth characteristics and cell cycle analysis after MNNG treatment were ascertained in seven MMR-deficient and proficient cell lines, with and without confirmed mutations in hMLH1 or hMSH2 by an in vitro transcription/translation assay. MMR-proficient cells underwent growth arrest in the G2 phase of the cell cycle after the first S phase, whereas MMR-deficient cells escaped an initial G2 delay and resumed a normal growth pattern. In the HCT116 line corrected for defective MMR by chromosome 3 transfer, the G2 phase arrest lasted more than five days. In another MMR-proficient colon cancer cell line, SW480, cell death occurred five days after MNNG treatment. A competent MMR system appears to be necessary for G2 arrest or cell death after alkylation damage, and this cell cycle checkpoint may allow the cell to repair damaged DNA, or prevent the replication of mutated DNA by prohibiting clonal expansion.

Alkylating Agents↗
PubMed1996

Loss of DNA mismatch repair in acquired resistance to cisplatin.

Selection of cells for resistance to cisplatin, a well-recognized mutagen, could result in mutations in genes involved in DNA mismatch repair and thereby to resistance to DNA-alkylating agents. Parental cells of the human ovarian adenocarcinoma cell line 2008 expressed hMLH1 when analyzed with immunoblot. One subline selected for resistance to cisplatin (2008/A) expressed no hMLH1, whereas another (2008/C13*5.25) expressed parental levels. Microsatellite instability was readily demonstrated in 2008/A cells but not in 2008 and in 2008/C13*5.25 cells. In addition, the 2008/A cells were 2-fold resistant to methyl-nitro-nitrosoguanidine and had a 65-fold elevated mutation rate at the HPRT locus as compared to 2008 cells, both of which are consistent with the loss of DNA mismatch repair in these cells. To determine whether the loss of DNA mismatch repair itself contributes to cisplatin resistance, studies were carried out in isogenic pairs of cell lines proficient or defective in this function. HCT116, a human colon cancer cell line deficient in hMLH1 function, was 2-fold resistant to cisplatin when compared to a subline complemented with chromosome 3 and expressing hMLH1. Similarly, the human endometrial cancer cell line HEC59, which expresses no hMSH2, was 2-fold resistant to cisplatin when compared to a subline complemented with chromosome 2 that expresses hMSH2. Therefore, the selection of cells for resistance to cisplatin can result in the loss of DNA mismatch repair, and loss of DNA mismatch repair in turn contributes to resistance to cisplatin.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Transcription-coupled repair deficiency and mutations in human mismatch repair genes.

Deficiencies in mismatch repair have been linked to a common cancer predisposition syndrome in humans, hereditary nonpolyposis colorectal cancer (HNPCC), and a subset of sporadic cancers. Here, several mismatch repair-deficient tumor cell lines and HNPCC-derived lymphoblastoid cell lines were found to be deficient in an additional DNA repair process termed transcription-coupled repair (TCR). The TCR defect was corrected in a mutant cell line whose mismatch repair deficiency had been corrected by chromosome transfer. Thus, the connection between excision repair and mismatch repair previously described in Escherichia coli extends to humans. These results imply that deficiencies in TCR and exposure to carcinogens present in the environment may contribute to the etiology of tumors associated with genetic defects in mismatch repair.

Adenosine Triphosphatases↗
PubMed1996

Microsatellite instability in nonneoplastic mucosa from patients with chronic ulcerative colitis.

Microsatellite instability (MIN) has been detected in many cancer types; however, recently we also observed it in the nonneoplastic but inflammatory setting of pancreatitis. Consequently, we sought to examine whether MIN was present in another inflammatory condition, ulcerative colitis (UC). MIN was found in 50% of UC patients whose colonic mucosa was negative for dysplasia, 46% of those with high-grade dysplasia, and 40% of those with cancer but in none of the ischemic or infectious colitis controls (P<0.03). Thus, UC patients may have MIN within mucosa that has no histological evidence of neoplastic change. MIN in this setting may reflect the inability of DNA repair mechanisms to compensate for the stress of chronic inflammation, and may be one mechanism for the heightened neoplastic risk in UC.

Colitis, Ulcerative↗
PubMed1996

Roles of the DNA mismatch repair genes in colorectal tumorigenesis.

A rapid increase in our understanding of the basic mechanisms involved in the genesis of colorectal cancer has occurred. Important factors include the activation of oncogenes, the inactivation of tumor suppressor genes and the inactivation of DNA repair systems. Hereditary non-polyposis colorectal cancer (HNPCC) has been linked to germ-line mutations that inactivate one allele of a DNA mismatch repair gene, which pre-disposes affected individuals to a high frequency of early-onset cancer. We have developed a cellular model to study HNPCC using the DNA mismatch repair-deficient colon cancer cell line HCT116 and corrected the genetic deficiency by chromosome transfer. Human chromosome 3 corrected all manifestations of DNA mismatch repair deficiency in the cell line and restored sensitivity to the toxic effects of certain DNA alkylating agents. Data suggest that the DNA mismatch repair genes not only participate in the recognition and repair of DNA mismatches during new strand synthesis but may also signal cell cycle arrest in the face of overwhelming DNA damage. Additional functions for the DNA mismatch repair genes in the maintenance of DNA replicative fidelity are being studied.

Alleles↗
PubMed1996

DNA repair and colorectal cancer.

The mismatch repair system plays a major role in the processing of recombination intermediates and in the repair of errors made during DNA replication or resulting from chemical damage to DNA. Human homologues of the bacterial and yeast mismatch repair genes have been recently identified, and mutations in these genes have been found to show risk for tumor development in hereditary nonpolyposis colorectal cancer syndrome (HNPCC). Colorectal tumors bearing homozygous mutations in these mismatch repair genes show a hypermutable phenotype, mainly at microsatellite regions of DNA. The temporal relationship between the loss of mismatch repair activity and the cascades of mutations in critical genes involved in the carcinogenesis of HNPCC tumors is unknown.

Animals↗
PubMed1996

Increased gastroduodenal concentrations of transforming growth factor alpha in adaptation to aspirin in monkeys and rats.

BACKGROUND & AIMS: The mechanism by which gastric mucosa becomes more resistant to damage by repeated aspirin administration is not known. Transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) prevent drug-induced gastric injury. The aim of this study was to determine whether gastroduodenal tissue levels of TGF-alpha and EGF protein were altered during adaptation to aspirin-induced injury in monkeys and rats in vivo. METHODS: Animals were given aspirin daily for up to 28 days. Gross mucosal injury was assessed by computerized image analysis in rats and by endoscopy in monkeys. Mucosal concentrations of TGF-alpha and EGF were quantitated by radioimmunoassays from endoscopic biopsy samples in monkeys and from scraped mucosa in rats. RESULTS: Long-term administration of aspirin caused a significant increase in gastric and duodenal tissue levels of TGF-alpha in monkeys and rats; the increased levels of TGF-alpha significantly correlated with the decrease in aspirin-induced injury. No change in the gastroduodenal tissue levels of EGF was observed. Adaptation was not associated with any significant change in basal gastric acid secretion in monkeys and occurred despite a significant decrease in gastric mucin in rats. CONCLUSIONS: Adaptation of the gastric mucosa to the damaging effect of aspirin is associated with a significant and specific increase in TGF-alpha protein in the gastroduodenum.

Adaptation, Physiological↗
PubMed1996

The effects of omeprazole and famotidine on mucin and PGE2 release in the rat stomach.

BACKGROUND: Gastric antisecretory agents may inhibit the synthesis or secretion of gastric mucin during acid suppression, which would interfere with mucosal protection and limit the efficacy of the agents. METHODS: Rats were dosed with famotidine, omeprazole, or buffer control for 4 weeks. Mucin synthesis, mucin histochemistry, mucin carbohydrate composition and prostaglandin E2 (PGE2) release were measured during and after drug treatment. RESULTS: PGE2 release was maximally inhibited after 2 weeks of omeprazole or 4 weeks of famotidine. Total glycoprotein synthesis was inhibited at all times by omeprazole, but only after the cessation of dosing with famotidine. Sulphated glycoprotein synthesis was inhibited by both drugs at 2 weeks. PGE2 release and sulphated glycoprotein synthesis were restored to control values or more by the 5th day after the end of dosing, at which time total glycoprotein synthesis was significantly suppressed in both groups. Histologically, a reduction of PAS-positive surface mucus was observed after 2 weeks of dosing in both groups. Both famotidine and omeprazole reduced the sialic acid content during and after treatment. CONCLUSIONS: These results suggest that long-term anti-secretory therapy also affects the production of factors involved in primary gastric mucosal defence, which should be considered in the assessment of response to treatment in clinical trials.

Animals↗
PubMed1995

Evidence for a connection between the mismatch repair system and the G2 cell cycle checkpoint.

The human colon tumor cell line HCT116 is deficient in wild-type hMLH1, is defective in mismatch repair (MMR), exhibits microsatellite instability, and is tolerant to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Transferring a normal copy of hMLH1 on chromosome 3 into the cell line restores MMR activity, stabilizes microsatellite loci, and increases the sensitivity of the cell to MNNG. Previous studies in other cell lines tolerant to alkylating agents such as MNNG or N-methylnitrosourea have shown cross-tolerance to 6-thioguanine (6TG), leading to a hypothesis that tolerance to MNNG or 6TG may be the result of MMR deficiency. To test this hypothesis, we studied the effects of 6TG on the MNNG-tolerant, MMR-deficient HCT116 cell line and its MNNG-sensitive, MMR-proficient, MNNG-tolerant, and MMR-deficient derivatives. Continuous exposure to low doses of 6TG (0.31-1.25 micrograms/ml) had no apparent effect on colony-forming ability (CFA) in MNNG-tolerant, MMR-deficient cells, whereas MNNG-sensitive, MMR-proficient cells exhibited a dose-dependent decrease in CFA. Growth kinetics and cell cycle analysis revealed that the growth of 6TG-treated HCT116 + chr3 cells was arrested at G2 after exposure to low dose of 6TG. In contrast, the same exposure to 6TG did not induce G2 arrest but rather a G1 delay in HCT116 and HCT116 + chr2. To obtain further evidence for the role of MMR on 6TG and MNNG toxicity, we isolated an MNNG-resistant revertant clone, M2, from the MNNG-sensitive, MMR-proficient HCT116 + chr3 cell line and characterized the MMR activity, hMLH1 status, and 6TG response. The results showed that M2 cells lost MMR activity as well as the previously introduced normal hMLH1 gene. Restoration of the CFA of M2 and an absence of G2 arrest were observed after treatment with low doses of 6TG. These results suggest that the mismatch repair system interacts with the G2 checkpoint in response to 6TG or MNNG-induced DNA lesions. The results further suggest that any agent that induces DNA mispairs will cause G2 arrest in MMR-proficient cells but not in MMR-deficient cells.

Azaguanine↗
PubMed1995

Hereditary nonpolyposis colorectal cancer: the syndrome, the genes, and historical perspectives.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disorder characterized by the occurrence within a family of multiple cases of colorectal cancer in the absence of gastrointestinal polyposis. The prevalence of this syndrome is not yet clear, but it may account for 1%-5% of all colorectal cancers. Prior to the identification of the genetic basis of this syndrome, the disease was recognized by the familial aggregation of colorectal cancers that had an early age of onset, an excess of proximally located, and often multiple, primary tumors, and an excess occurrence of cancers in certain other organs. The recent description of an abnormality called "microsatellite instability," present in almost all cancers from HNPCC patients and in about 12%-15% of sporadic cases, led to a series of discoveries that linked this type of genomic instability to a defect in the DNA mismatch repair (MMR) system. Independent investigators have identified four HNPCC genes: hMSH2 (a homologue of the prokaryotic DNA MMR gene MutS) and hMLH1, hPMS1, and hPMS2 (all homologues of the prokaryotic DNA MMR gene MutL). Mutations in each of the four genes have been found in the germline cells of HNPCC families. A major target for research in this area is the development of clinically practical screening tests for the genetic carrier state of HNPCC.

Colorectal Neoplasms, Hereditary Nonpolyposis↗
PubMed1995

Microallelotyping defines the sequence and tempo of allelic losses at tumour suppressor gene loci during colorectal cancer progression.

Microallelotyping of many regions from individual colorectal tumours was used to determine the sequence and tempo of allelic loss on 5q, 17p and 18q during neoplastic progression. No allelic losses were found in normal tissues surrounding colorectal neoplasms, but losses occurred abruptly on 5q at the transition from normal colonic epithelium to the benign adenoma, and on 17p at the transition from adenoma to carcinoma, indicating an essential role for these losses in tumour progression. Allelic losses were uniform throughout extensively microdissected benign adenomas and carcinomas. However, substantial allelic heterogeneity was found in high-grade dysplasia, the transition lesion between adenoma and carcinoma. Thus, allelic losses on 5q and 17p are associated with abrupt waves of clonal neoplastic expansion, and high-grade dysplasia is characterized by a high degree of allelic heterogeneity.

Adenocarcinoma↗
PubMed1994

Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability and reduces N-methyl-N'-nitro-N-nitrosoguanidine tolerance in colon tumor cells with homozygous hMLH1 mutation.

The human colon tumor cell line HCT 116 is known to have a homozygous mutation in the mismatch repair gene hMLH1 on human chromosome 3, to exhibit microsatellite instability, and to be defective in mismatch repair. In order to determine whether the introduction of a normal copy of hMLH1 gene restores mismatch repair activity and corrects microsatellite instability, a single human chromosome 3 from normal fibroblasts was transferred to HCT 116 cells via microcell fusion. As a control, human chromosome 2 was also transferred to HCT 116 cells. Two HCT 116 microcell hybrid clones that received a single copy of chromosome 2 (HCT 116 + ch2) and two that received a single copy of chromosome 3 (HCT 116 + ch3) were isolated and characterized. A G-G mismatch in M13-derived heteroduplex DNA was efficiently repaired in cell extracts from HCT 116 + ch3 cells, but not in those of parent HCT 116 cells or HCT 116 + ch2 cells. Microsatellite alterations at the D5S107 locus containing CA repeats were seen in 8 of 80 subclones from HCT 116 cells, and in 13 of 150 subclones from HCT 116 + ch2 cells. In contrast, none of the 225 subclones derived from mismatch repair-proficient HCT 116 + ch3 cells showed alterations in the microsatellite at the same locus. The effect of introducing chromosome 3 on the sensitivity of HCT 116 cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined, since enhanced tolerance to MNNG is accompanied by loss of mismatch repair activity in several cell lines. Within 3 days after treatment with 5 microM MNNG, HCT 116 + ch3 cells became morphologically flat and stopped growing. Their colony-forming ability, determined 10 days after treatment, was reduced 200-fold when compared to MNNG-treated parental HCT 116 and HCT 116 + ch2 cells. These results support the hypothesis that mutations in both alleles of the hMLH1 gene are necessary for the manifestation of defective mismatch repair and microsatellite instability and for enhanced MNNG tolerance. The results also suggest that the mismatch repair system contributes to the process that causes growth arrest in response to DNA damage by alkylating agents.

Base Sequence↗
PubMed1994

The lectin Griffonia simplicifolia I-A4 (GS I-A4) specifically recognizes terminal alpha-linked N-acetylgalactosaminyl groups and is cytotoxic to the human colon cancer cell lines LS174t and SW1116.

The lectin GS I-A4 binds to terminal alpha-N-acetylgalactosaminyl (GalNAc) groups (which include the Tn antigen), but not to the closely related tumor-associated epitope, sialylated Tn antigen. The lectin also precipitates asialo OSM, but not its native sialylated form. Lectin histochemistry with human colonic tissues showed that GS I-A4 specifically stained specimens of colon cancer and colonic tissues from individuals with FAP; however, normal colonic tissues from patients without colonic disease were rarely stained with this lectin. Glycoconjugates bound by GS I-A4 were observed on the surface membranes of 2 human colon cancer cell lines, LS174t and SW1116, when fluorescein isothiocyanate (FITC)-conjugated GS I-A4 was used. GS I-A4 was toxic to these 2 human colon cancer cell lines in monolayer culture. A dose-response study conducted using 10-160 micrograms/ml, of GS I-A4 demonstrated significant dose-related toxicity against LS174t and SW1116 cells. At concentrations > 80 micrograms/ml, > 99% of LS174t and > 90% of SW1116 cells were killed. Four mM GalNAc specifically inhibited the cytotoxic effect of GS I-A4 (p < 0.001), whereas 4mM N-acetylglucosamine (GlcNAc) had no effect. Two other lectins that recognize terminal alpha-GalNAc residues, DBA and LBL, were significantly less cytotoxic to the colon cancer cells than GS I-A4. In the light of these findings, we speculate that GS I-A4 may have potential use as a diagnostic agent against colorectal cancer.

Acetylgalactosamine↗