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PubMed2000

Dose-response relationship for radiation-induced mutations at micro- and minisatellite loci in human somatic cells in culture.

PURPOSE: The study was designed to determine the dose-response relationship for radiation induction of mutations at mini- and microsatellite loci in human somatic cells. Mutations induced by graded doses of gamma-irradiation were quantified by screening clones derived from single irradiated cells for micro- and minisatellite alterations following irradiation with 1, 2 or 3 Gy. MATERIALS AND METHODS: After irradiation, the moderately radioresistant glioma cell line UVW was seeded at low density into Petri dishes to allow formation of discrete colonies, 100 of which were examined at each dose. All the cells within a colony were presumed to have arisen from a single irradiated cell. Radiation-induced microsatellite alterations were determined at 16 different loci, by PCR amplification and visualization on polyacrylamide gels. Minisatellite alterations were identified at four different minisatellite loci by restriction enzyme digestion and Southern blotting. RESULTS: A dose-response curve for mutation frequency was obtained by analysis of 100 clones, yielding a minisatellite mutation rate of 5.5x10(-3) mutations/locus/Gy/cell and a microsatellite mutation rate of 8.75x10(-4) mutations/locus/ Gy/cell. At microsatellite loci, alterations were predominantly simple loss or gain of repeat units and loss of heterozygosity (LOH). The mutations in minisatellite loci resulted predominantly in LOH and variation in repeat number. The background instability at each locus was determined by analysis of non-irradiated clones. Only 2% and 1% of the micro-and minisatellite loci respectively showed altered bands. CONCLUSIONS: This is the first report of a dose-response relationship for radiation-induced micro- and minisatellite mutations in human somatic cells. Described is a sensitive method for analysis of low-dose radiation mutagenesis in somatic cells that may prove to be a useful tool for radiation protection and dosimetry.

Dose-Response Relationship, Radiation↗
PubMed1999

PMS2-deficiency diminishes hypermutation of a lambda1 transgene in young but not older mice.

The Pms2 gene is involved in DNA mismatch repair in mammalian cells, and has recently been shown to affect hypermutation of mammalian immunoglobulin genes. We have studied hypermutation of a lambda1 transgene in chronically stimulated Peyer's patch B cells of both young and old mice deficient in function of Pms2. In young (3-4 months) mice, somatic hypermutation is fourfold lower in PMS2-deficient mice than in control mice. This difference is statistically significant (P < 0.05). In contrast, in older mice (9 months of age), hypermutation levels are indistinguishable in the Pms2-/- and Pms2+/+ backgrounds. In the older mice, there was no clear difference in the fraction of clones carrying either any mutations or at least two mutations when PMS2-deficient mice were compared with their wild-type littermates. As genomic instability increases with age, this observation is difficult to reconcile with the hypothesis that highly mutated B cells cannot survive in Peyer's patches. Moreover, there were clear differences apparent in the mutation spectra of the Pms2-/- and Pms2+/+ mice. In the PMS2-deficient background, deletion and insertion mutations were found, and there was a significant decrease in the ratio of A mutations to T mutations in comparison with the Pms2+/+ controls. Our data support the hypothesis that PMS2 functions in somatic hypermutation, and are most consistent with the hypothesis that the role of PMS2 is direct rather than indirect.

Adenosine Triphosphatases↗
PubMed2006

A decerebrate, artificially-perfused in situ preparation of rat: utility for the study of autonomic and nociceptive processing.

By extending established technology, we have developed a relatively simple in situ preparation from juvenile rat that overcomes some technical restrictions present in vivo (e.g. need for anaesthesia, mechanical instability for intracellular recording and control over the extracellular milieu). The in situ preparation is decerebrate and artificially perfused via the left ventricle with a colloid containing solution. It exhibits an eupneic pattern of respiratory motor activity and demonstrates numerous somatic and visceral reflexes including those evoked by stimulation of the tail, hindlimbs, bladder, baroreceptors and peripheral chemoreceptors. We have employed this preparation to allow recordings from multiple sympathetic motor outflows such as thoracic and lumbar chain, inferior cardiac, splanchnic, renal and adrenal nerves. We show that the sympathetic motor discharge shows strong respiratory modulation and exhibits pronounced reflex modulation indicating intact communication between the periphery, the brainstem and the spinal cord. Further, we have made extracellular and whole cell recordings from neurones in the spinal cord, demonstrating good mechanical stability. The decerebrate, artificially-perfused rat (DAPR) provides a powerful methodology with which to study peripheral and central control of the autonomic nervous system with many of the benefits of an in vitro environment.

Action Potentials↗
PubMed2001

Ever since Knudson.

Many publications have documented loss of heterozygosity (LOH) on many different chromosomes in a wide variety of tumours, implicating the existence of multiple tumour suppressor genes (TSGs). Knudson's two-hit hypothesis predicts that these LOH events are the second step in the inactivation of both alleles of a TSG. However, to date the number of TSGs identified that are inactivated mainly at the somatic level in cancers and are not inherited has remained disappointingly small. Here we postulate that the accurate mapping of LOH events in a series of tumours to define a common LOH region is greatly confounded by deficient LOH detection, genetic instability and intertumour heterogeneity. Finding the TSGs in chromosomal regions of frequent LOH might require 'brute-force' genomic approaches.

Alleles↗
PubMed2002

Adverse effects of pain on the nervous systems of newborns and young children: a review of the literature.

There are immediate and long-lasting harmful consequences to the nervous system when infants experience severe or repetitive pain. These effects are especially significant in preterm infants, who are vulnerable to neurological damage during this critical time of neurodevelopment. Painful experiences may cause structural and physiological changes within the nervous system. Repeated painful procedures may result in decreased pain thresholds and hypersensitivity to pain. Immediate harmful effects of pain include physiologic instability and increased incidence of serious complications such as intraventricular hemorrhage. Painful stressors may lead to sleep disturbances, feeding problems, and inability to self-regulate. Long-term effects of pain may include altered pain perception, chronic pain syndromes, and somatic complaints. Repetitive pain in the preterm infant may be associated with attention deficit disorders, learning disorders, and behavioral problems in later childhood. Nursing involvement with pain management is crucial to achieve positive health outcomes for high-risk infants and older children and adults who have experienced repetitive or severe pain as infants.

Humans↗
PubMed1996

Frequent microsatellite instabilities and analyses of the related genes in familial gastric cancers.

Microsatellite instability or replication error seems to be related to defective DNA mismatch repair genes, such as hMSH2, hMLH1, hPMS1 and hPMS2, which have been identified as causative genes of hereditary nonpolyposis colorectal cancers (HNPCC). Recently, it was reported that mutations at the simple repeated sequences in the transforming growth factor-beta type II receptor (TGF-beta RII) gene occurred in replication error-positive colorectal cancers. To determine genetic alterations in familial gastric cancers (FGC, we examined replication error using eight microsatellite DNA markers, and screened mutations in the hMSH2, hMLH1 and TGF-beta RII genes in six cases from four FGC kindreds. Moreover, hMTH1, a human homolog of the bacterial mutT gene, was also screened. Four of six (67%) cancers showed the replication error-positive phenotype, indicating that microsatellite instability is highly associated with not only HNPCC, but also FGC. No germline mutation was found in the whole coding sequences of hMSH2 and hMTH1, or in the conservative regions of hMLH1 in any patient, while one cancer DNA showed a somatic mutation at codon 682 (threonine to alanine) in hMSH2. No alteration was found at the small repeated sequences in TGF-beta RII in FGC tumor DNA. These results indicate that the carcinogenetic process of FGC may be different from that of HNPCC.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Gonosomal mosaicism in myotonic dystrophy patients: involvement of mitotic events in (CTG)n repeat variation and selection against extreme expansion in sperm.

Myotonic dystrophy (DM) is caused by abnormal expansion of a polymorphic (CTG)n repeat, located in the DM protein kinase gene. We determined the (CTG)n repeat lengths in a broad range of tissue DNAs from patients with mild, classical, or congenital manifestation of DM. Differences in the repeat length were seen in somatic tissues from single DM individuals and twins. Repeats appeared to expand to a similar extent in tissues originating from the same embryonal origin. In most male patients carrying intermediate- or small-sized expansions in blood, the repeat lengths covered a markedly wider range in sperm. In contrast, male patients with large allele expansions in blood (> 700 CTGs) had similar or smaller repeats in sperm, when detectable. Sperm alleles with > 1,000 CTGs were not seen. We conclude that DM patients can be considered gonosomal mosaics, i.e., combined somatic and germ-line tissue mosaics. Most remarkably, we observed multiple cases where the length distributions of intermediate- or small-sized alleles in fathers' sperm were significantly different from that in their offspring's blood. Our combined findings indicate that intergenerational length changes in the unstable CTG repeat are most likely to occur during early embryonic mitotic divisions in both somatic and germ-line tissue formation. Both the initial CTG length, the overall number of cell divisions involved in tissue formation, and perhaps a specific selection process in spermatogenesis may influence the dynamics of this process. A model explaining mitotic instability and sex-dependent segregation phenomena in DM manifestation is discussed.

Aged↗
PubMed2005

A single-molecule PCR approach to the measurement of induced expanded simple tandem repeat instability in vitro.

Sensitive and precise models are needed to identify potential genotoxicity at environmentally relevant doses of mutagens. The size length alterations in expanded simple tandem repeat (ESTR) loci have been used as a biomarker of genetic instability caused by a variety of agents in the mouse germline. The mechanisms operating in both spontaneous and induced instability are poorly understood. We have developed a single-molecule polymerase chain reaction (SM-PCR) method to investigate mutation at the mouse ESTR locus Ms6-hm in the murine C3H/10T1/2 embryonic cell line. Growth of cells to levels of high cell density induced increased ESTR instability, with mutation frequencies 5.1-fold (+/-2.8) over sub-confluent cultures. Accordingly, cell cultures were maintained at sub-confluent levels for further investigations of the induction of ESTR mutation by genotoxic agents. Treatment with the DNA alkylating agent N-nitroso-N-ethylurea (ENU) resulted in a 1.94-fold (+/-1.1) increase in mutation frequency, similar to responses measured previously in the germline in vivo. Therefore, mutagen exposure can also affect somatic (non-meiotic) rapidly dividing mouse cells. This SM-PCR approach eliminates the requirement of sub-cloning individual treated cells, thereby, reducing the time needed to screen for ESTR mutation, and will be a very useful tool for future investigations into the mechanisms involved in ESTR mutation.

Alleles↗
PubMed1992

High frequency, heat treatment-induced inactivation of the phosphinothricin resistance gene in transgenic single cell suspension cultures of Medicago sativa.

One descendant of the Medicago sativa Ra-3 transformant T304 was analysed with respect to the somatic stability of the synthetic phosphinothricin-N-acetyltransferase (pat) gene which was used as a selective marker and was under the control of the 5'/3' expression signals of the cauliflower mosaic virus (CaMV) gene VI. In order to quantify gene instability, we developed a system for culturing and regenerating individual cells. Single cell suspension cultures derived from T304 and the ancestral non-transgenic M. sativa cultivar Ra-3, were established. The cells were regenerated into monoclonal calli. In transgenic calli, the phosphinothricin (Pt)-resistance phenotype was retained after more than 2 months of non-selective growth. In contrast, up to 12% of the suspension culture cells grown under nonselective conditions and at constant temperature (25 degrees C) lost the herbicide-resistance phenotype within 150 days. Surprisingly, a heat treatment (37 degrees C), lasting for 10 days, during the culture period resulted in an almost complete (95%) loss of the Pt resistance of the suspension culture cells. However, the frequency of cell division was identical in cultures grown under normal and heat treatment conditions. A biochemical test revealed that no phosphinothricin-N-acetyltransferase activity was present in heat treated, Pt-sensitive cells. The resistance level of the Pt-sensitive transgenic cells was equivalent to that of the wild-type cells. A PCR analysis confirmed the presence of the pat gene in heat treated, Pt-sensitive cells. From these results it is concluded that the Pt resistance gene was heat-inactivated at a high frequency in the M. sativa suspension cultures.

Acetyltransferases↗
PubMed2002

Application of inter-simple sequence repeat PCR to mouse models: assessment of genetic alterations in carcinogenesis.

Genomic instability is believed to play a significant role in cancer development by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (inter-SSR) PCR has been proved to be a fast and reproducible technique for quantitation of genomic instability (amplifications, deletions, translocations, and insertions) in human sporadic tumors. However, the use of inter-SSR PCR in animal models of cancer has never been described. This new technique has been adapted in our laboratory for the analysis of spontaneous and induced mouse tumors. We established the best PCR conditions for each microsatellite-anchored primer and critically evaluated the reproducibility of the band patterns. We also studied the variation of the fingerprints between and within various inbred mouse strains, including wild-derived lines. Tumor-specific alterations were detected as gains, losses, or intensity changes in bands when compared with matched normal DNA. We quantitated the extent of alterations by dividing the number of altered bands in the tumor by the total number of bands in normal DNA (instability index). By means of inter-SSR PCR, we successfully analyzed genomic alterations in various mouse tumors, including spontaneous thymic lymphomas developed in Msh2 knockout mice as well as chemically induced squamous cell carcinomas and thymic lymphomas. Instability index values ranged between 0 and 9%, the highest levels observed in N-methyl-N-nitrosourea-induced thymic lymphomas generated in Trp53 (p53) nullizygote (-/-) mice. We report here, for the first time, the use of inter-SSR PCR to detect somatic mutations in mouse tumoral DNA, including laser-capture microdissected, methanol-fixed tissues. These PCR-based fingerprints provide a novel approach to assessing the number and onset of mutational events in mouse tumors and will help to understand better the mechanisms of carcinogenesis in mouse models.

Animals↗
PubMed2000

Genetic aberration in primary hepatocellular carcinoma: correlation between p53 gene mutation and loss-of-heterozygosity on chromosome 16q21-q23 and 9p21-p23.

To elucidate the molecular pathology underlying the development of hepatocellular carcinoma (HCC), we used 41 highly polymorphic microsatellite markers to examine 55 HCC and corresponding non-tumor liver tissues on chromosome 9, 16 and 17. Loss-of-heterozygosity (LOH) is observed with high frequency on chromosomal region 17p13 (36/55, 65%), 9p21-p23 (28/55, 51%), 16q21-q23 (27/55, 49%) in tumors. Meanwhile, microsatellite instability is rarely found in these microsatellite loci. Direct sequencing was performed to detect the tentative mutation of tumor suppressor genes in these regions: p53, MTS1/p16, and CDH1/E-cadherin. Within exon 5-9 of p53 gene, 14 out of 55 HCC specimens (24%) have somatic mutations, and nucleotide deletion of this gene is reported in HCC for the first time. Mutation in MTS1/p16 is found only in one tumor case. We do not find mutations in CDH1/E-cadherin. Furthermore, a statistically significant correlation is present between p53 gene mutation and loss of chromosome region 16q21-q23 and 9p21-p23, which indicates that synergism between p53 inactivation and deletion of 16q21-q23 and 9p21-p23 may play a role in the pathogenesis of HCC.

Cadherins↗
PubMed2002

Effectiveness of adjunctive gabapentin in resistant bipolar disorder: is it due to anxious-alcohol abuse comorbidity?

We evaluated effectiveness and predictors of response of gabapentin (GBP) as adjunctive treatment in a sample of 43 subjects with DSM-III-R bipolar disorder who were resistant to standard mood stabilizers. Diagnostic evaluation was performed by means of the Semistructured Interview for Mood Disorder. Clinical evaluation was performed at the beginning and end of the observation period by means of the Hamilton Rating Scale for Depression (HAM-D), the Young Mania Rating Scale, and the Clinical Global Impression Scale. GBP was administered as an adjunctive treatment for an 8-week period in combination with other mood stabilizers, benzodiazepines, antidepressants, and neuroleptics. Mean dosage +/- SD at week 8 was 1270 +/- 561.4 mg (range, 600-2400 mg). Adjunctive treatment with GBP was well tolerated by almost all the subjects; only three patients had to interrupt treatment before week 8, two because of inefficacy and one because of the appearance of side effects (ataxia and irritability); in other patients, the most frequent side effects were sedation, irritability, tremor, ataxia or motor instability, and nausea. Eighteen (41.9%) of 43 patients who began treatment were considered responders. Mean total HAM-D score showed a significant reduction during the 8 weeks of treatment. Analysis of the various HAM-D dimensions showed that the anxiety-somatization factor was the one with the greatest change. Seventeen of the 18 responder patients remained in remission for a period ranging from 4 to 12 months without clinically significant side effects or adverse events. One patient had to interrupt GBP treatment and be administered neuroleptics because of the reappearance of manic symptoms. Regarding response predictors, logistical regression analysis showed that the presence of panic disorder and alcohol abuse was associated with positive response. The results of the present study replicate prior studies indicating that GBP is an effective and well tolerated treatment in a large proportion of bipolar patients who are resistant to traditional mood stabilizers. More specifically, this drug appears to have antidepressant and anxiolytic properties. What is new in the present report is the suggestion that the utility of GBP in resistant bipolar disorder resides in its effectiveness against comorbid panic disorder and alcohol abuse.

Acetates↗
PubMed1996

Allelic deletion on chromosome 17p13.3 in early ovarian cancer.

Multiple chromosome 17 loci may be involved in ovarian carcinogenesis. Fifty-seven sporadic ovarian epithelial tumors were examined for loss of heterozygosity at 15 loci on chromosomes 17p. Eighty % (39 of 49) of informative tumors had allelic loss in 17p13.3 at D17S30, D17S28, or both loci within this region, including 3 of 7 tumors of low malignant potential and 4 of 5 nonmetastatic carcinomas. The smallest region of overlapping deletions extends from D17S28 to D17S30, a distance of 15 kb. Furthermore, several tumors have breakpoints within the region detected by the D17S30 probe. Chromosome 17p13.3 genes with potential tumor suppressor function include HIC-1, DPH2L (N. J. Phillips et al. Isolation of a human diphthamide biosynthesis gene on chromosome 17p13.3, submitted for publication)/OVCA1, PEDF, and CRK. The HIC-1 coding sequence lies i kb centromeric to the D17S28-S17S30 region of deletion (M. Makos Wales et al., Nat. Med., 1:570-577, 1995) but remains a candidate because 5'-regulatory elements may lie within the critical region. Portions of the DPH2L/OVCA1 coding sequence lie within the D17S28-D17S30 interval. Somatic cell hybrid analysis places PEDF in an interval including D17S28, D17S30, and D17S54, whereas CRK is excluded from this interval. Chromosome 17p13.3 loss precedes TP53 and BRCA1 region deletions because the latter changes are see only in high-stage carcinomas. Microsatellite instability plays only a minor role in sporadic ovarian carcinogenesis because only 1 of 57 tumors showed this finding.

Adult↗
PubMed1996

Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia.

Fanconi anaemia (FA) is an autosomal recessive disease characterized by bone marrow failure, variable congenital malformations and predisposition to malignancies. Cells derived from FA patients show elevated levels of chromosomal breakage and an increased sensitivity to bifunctional alkylating agents such as mitomycin C (MMC) and diepoxybutane (DEB). Five complementation groups have been identified by somatic cell methods, and we have cloned the gene defective in group C (FAC)(7). To understand the in vivo role of this gene, we have disrupted murine Fac and generated mice homozygous for the targeted allele. The -/- mice did not exhibit developmental abnormalities nor haematologic defects up to 9 months of age. However, their spleen cells had dramatically increased numbers of chromosomal aberrations in response to MMC and DEB. Homozygous male and female mice also had compromised gametogenesis, leading to markedly impaired fertility, a characteristic of FA patients. Thus, inactivation of Fac replicates some of the features of the human disease.

Animals↗
PubMed2005

Chromosomal instability predicts metastatic disease in patients with insulinomas.

Endocrine pancreatic tumors (EPTs) comprise a highly heterogeneous group of tumors with different clinical behavior and genetic makeup. Insulinomas represent the predominant syndromic subtype of EPTs. The metastatic potential of insulinomas can frequently not be predicted using histopathological criteria, and also molecular markers indicating malignant progression are unreliable because of the small number of cases per subtype studied so far. For the identification of reliable indicators of metastatic disease, we investigated 62 sporadic insulinomas (44 benign and 18 tumors with metastases) by means of comparative genomic hybridization (CGH). In addition, the role of MEN1 (multiple endocrine neoplasia type 1) gene mutations was determined to assess specific chromosomal alterations associated with dysfunction of this endocrine tumor-related tumor suppressor gene. Only one case with a somatic MEN1 mutation was identified (1527del7bp), indicating that the MEN1 gene plays a minor pathogenic role in sporadic insulinomas. CGH analysis revealed that the total number of aberrations per tumor differs strongly between the benign and the malignant group (4.2 vs 14.1; P<0.0001). Furthermore, chromosome 9q gain was found to be the most frequent aberration in both benign and malignant insulinomas, whereas chromosome 6q losses and 12q, 14q and 17pq gains are strongly associated with metastatic disease. Our study shows that chromosomal instability, as defined by > or =5 gains together with > or =5 losses, or total number of gains and losses > or =8, rather than parameters such as tumor size and proliferation index, is the most powerful indicator for the development of metastatic disease in patients with sporadic insulinoma.

Adult↗
PubMed1997

alpha-satellite DNA methylation in normal individuals and in ICF patients: heterogeneous methylation of constitutive heterochromatin in adult and fetal tissues.

The methylation profile of ten alpha-satellites was investigated in normal individuals and in ICF (Immunodeficiency, Centromeric instability, Facial abnormalities) patients. Two out of three ICF patients showed modified methylation of these sequences, reproducing a placental profile. CENP-B boxes, the binding sites of centromeric protein B, were always skewed toward nonmethylation. Unexpected results were observed in normal individuals: in somatic adult tissues the methylation pattern of alpha-satellite DNA varied between chromosomes, and in fetal tissues these satellites were homogeneously undermethylated. Detailed methylation analysis of CENP-B boxes revealed that unmethylated alpha-satellite units coexist with thoroughly methylated regions. These observations showed that the two major components of constitutive heterochromatin are differently methylated in normal somatic and fetal tissues, since classical satellites are consistently methylated. The definite changes in the methylation profile of heterochromatin in somatic chromosomes and the asynchronous timing of methylation of classical and alpha-satellites during development may reflect specific roles of highly repeated sequences in genomic organization.

Adolescent↗
PubMedDate not supplied

Molecular genetics of hereditary non-polyposis colorectal cancer (HNPCC).

The story of the molecular genetics of HNPCC is one of astonishingly rapid achievements. In just 16 months, from May 1993 to September 1994, four different genes, namely hMSH2, hMLH1, hPMS1 and hPMS2 have been identified and demonstrated to be associated with the disease. Their cloning was facilitated by the finding that tumor cells in HNPCC patients display a hypermutability of DNA short tandem repeats (microsatellite instability). In fact, HNPCC associated genes are the human counterparts of genetic elements known to control the fidelity of DNA replication in lower organisms. So far, more than 50 germline mutations of hMSH2 and hMLH1 genes have been reported in HNPCC kindreds. In addition, somatic mutations have been documented in hereditary as well as sporadic cancers. Unfortunately, the molecular diagnosis of HNPCC is hampered by the lack of mutational "hot spots" and of clearly defined genotype-phenotype correlations and different screening methods are to be employed for the analysis of affected and at-risk individuals.

Colorectal Neoplasms, Hereditary Nonpolyposis↗