ABSTRACT
The analysis of recurring chromosome aberrations has become an integral part of the diagnostic and prognostic workup of many human cancers, and their molecular analyses have facilitated the identification of genes related to the pathogenesis of cancer. Cholangiocarcinoma (CC), a malignant neoplasm of the biliary epithelium, is usually fatal because of the difficulty in early diagnosis and unavailability of effective therapy. Furthermore, little is known about the genetics and biology of CC. Only few reports concerning cytogenetic studies of CC have been published and few cell lines have been established. We recently established CC cell line, designated as PCK1. The purpose of this study is to establish in detail karyotype of PCK1 cell line. The origins of the unidentified marker chromosomes were analyzed by G-banding, cross species color banding (RxFISH), and human chromosome-specific painting. In PCK1 cell line, gains involved chromosomes and chromosome regions, 4, 5, 9, 12, 16, 21, 1q, 7q11-q22, 8q, 12p, 14q11-q22, 15q21-qter, 17p11-qter, and 18p. Losses involved Y, 7q31-qter, 8p, 14q23-qter, 17p12-pter, and 18q. Established PCK1 cell line will be able to use the basic research of cholangiocarcinoma and the abnormal chromo-somes may be the candidate regions for isolation of the genes related to CC.
Subject(s)
Humans , Biology , Cell Line , Cholangiocarcinoma , Chromosome Aberrations , Cytogenetics , Early Diagnosis , Epithelium , Genetics , Karyotype , Paint , PaintingsABSTRACT
In this study, S59158, a gene of GLAST (L-Glutamate/L-Aspartate transporter), was cloned by ordered differential display PCR with developing rat brains. The mRNA expression of this gene in the developing rat brain and the effect of kainic acid (KA), glutamate analogue, on this gene were investigated with in situ hybridization histochemistry. The expression of S59158 was restricted to nervous system and observed from E12 (embryonic day 12), peaked at E20, and gradually decreased to adult level. In embryos, S59158 was prominently expressed in the subventricular zones throughout the brain. After birth, strong expression was observed in the purkinje cell layer of cerebellum and moderate level of expression was observed in the subventricular zone, olfactory bulb, hippocampal formation, and cerebral cortex. In the KA treated rat brains, the expression of S59158 was significantly increased in dentate gyrus, hippocampus, and cerebral cortex. From these results, it may be suspected that S59158 is related to the development of the brain and is induced by increased extracellular glutamate level.
Subject(s)
Adult , Animals , Humans , Rats , Amino Acid Transport System X-AG , Brain , Cerebellum , Cerebral Cortex , Clone Cells , Cloning, Organism , Dentate Gyrus , Embryonic Structures , Genes, vif , Glutamic Acid , Hippocampus , In Situ Hybridization , Kainic Acid , Nervous System , Olfactory Bulb , Parturition , Polymerase Chain Reaction , RNA, MessengerABSTRACT
PURPOSE: Chromosome microdissection has become a very powerful approach to generate chromosome band-specific library and painting probes for physical mapping or cytogenetic analysis. We have constructed here band-specific painting probes for human chromosomes by microdissection and polymerase chain reaction(PCR). METHODS: We pretreated the microdissected fragments with Topoisomerase I(Topo I) which catalyzes the relaxation of supercoiled DNA, and performed two initial rounds of DNA synthesis with T7 DNA polymerase followed by conventional PCR to enable the reliable preparation of fluorescent in situ hybridization(FISH) probe. RESULTS: It was possible to construct region-specific painting probes for 5q14-q15, 6q23, 16p11.2 and 22q12. The painting probes were successful in determining the corresponding chromosome bands in SNU-484 cell line, IMR-32 cell line. CONCLUSION: These painting probes can be used to detect the related marker chromosomes in congenital malformations and other solid tumors, and the band specific probe pools may be used effectively to analyse the genes.
Subject(s)
Humans , Cell Line , Chromosomes, Human , Cytogenetic Analysis , DNA , DNA, Superhelical , Microdissection , Paint , Paintings , Polymerase Chain Reaction , RelaxationABSTRACT
PURPOSE: The characterization of all recognizable chromosomal rearrangements was dis- turbed by technical limitation of conventional cytogenetic methods. Recently, the strong usefullness of generation of chromosome specific painting probes in identification of marker chromosomes has proven. This study was intended to analyze the chromosomal aberrations in human ovarian cancer cell line, SNU-8, by G-banding and multiple paintings. MATERIALS AND METHODS: Human ovarian cancer cell line, SNU-8 was cultured and harvested for cytogenetic analysis. Routine karyotyping was performed. For complete analysis of chromosomal aberrations, human chromosome-specific painting probes were constructed from somatic hybrid cells. The origins of the unidentified marker chromosomes were analyzed by fluorescent in situ hybridization (FISH) with these painting probes. RESULTS: All chromosome alterations were confirmed by the use of multiple chromosome paintings, which also demonstrated a number of additional alterations. SNU-8 had the karyotype 62-69,XXX, + der(1;10)(q10;p10),der(3;18) (q10;p10)X2,-4,+ 5,+ 7,del(9)(q21)X2,-11,-13,-15,-16,der(17;19)(q10;q10) X2, + 20,-22[cp51]. CONCLUSION: The chromosomal aberrations of SNU-8 cell line was effectively analyzed by FISH with these painting probes, and the approach methods of this study can be applied to cytogenetic analysis of chromosomal aberrations in the other cancers.
Subject(s)
Humans , Cell Line , Cell Line, Tumor , Chromosome Aberrations , Chromosome Painting , Cytogenetic Analysis , Cytogenetics , Hybrid Cells , In Situ Hybridization, Fluorescence , Karyotype , Karyotyping , Ovarian Neoplasms , Paint , PaintingsABSTRACT
In rat that is helpless at birth, the cerebellum is in a corresponding state of immaturity, and its histogenesis and morphogenesis mainly occur after birth. The times and sites of origin of the four types of cerebellar local-circuit neurons, as well as their migration routes to specific positions in the cortex, their distinctive patterns of differentiation and growth, and their synaptogenesis, have been well studied. The stage-specific genes in the postnatal rat cerebellum may be related with these kind of neural development in the cerebellum. To clone the genes related with neural development in the postnatal cerebellum, developmentally differentially expressed genes were screened from postnatal rat cerebellum with ordered differential display (ODD) and the developmental expression pattern in the postnatal rat cerebella was investigated with in situ hybridization histochemistry. One novel postnatal stage-specific gene (PKrCb1) was cloned by ODD with 7 cDNA pools (P0, P3, P7, P12, P18, P25, adult rat cerebella). To investigate the developmental expression pattern of this novel gene on the cell level, in situ hybridization histochemistry was performed in the developing and adult rat brain sections. The developmental expression pattern of PKrCb1 in the cerebellum was well matched with spatiotemporal migration pattern of granule cells and it may be suspected that PKrCb1 is related with migration of granule cells from external granular layer to internal granular layer. From the results, it is suggested that the methods used in this experiment will be the powerful methods for the cloning and primary function study of the genes related with cerebellar development.
Subject(s)
Adult , Animals , Humans , Rats , Brain , Cerebellum , Clone Cells , Cloning, Organism , DNA, Complementary , In Situ Hybridization , Morphogenesis , Neurons , ParturitionABSTRACT
The tottering (tg/tg) is neurologic mutant mouse exhibiting three neurological disorders: ataxia, petit mal-like absence seizures and myoclonic intermittent movement disorder. The tottering mouse carries an autosomal recessive single gene mutation on chromosome 8. The leaner (tgla) and Nagoya rolling (tgrol) are another two alleles of the tottering (tg). The combination of two mutant (tottering and leaner) produces compound heterozygous, tottering/leaner (tg/tgla) mouse. The genetic etilogy of the tottering and leaner was identified to be a mutation in voltage-dependent calcium channel a1A subunit. It made us link these animal model to human neurologic disease such as autosomal dominant cerebellar ataxia (SCA6), familial hemiplegic migraine and episodic ataxia type-2. The different onset and severity of neurological symptom of these three mutants (tg/tg, tg/tgla, tgla/tgla) offer good scale to analysis of pathophysiolgy of the neurologic disorder. Altered synapase between parallel fiber varicosity and dendritic spines of Purkinje cell was observed in adult tottering and leaner mice. Through the electron microscopic observation and anticalbindin-28 kd immunohistochemistry, we anaylzed not only the relationship between neurologic symptoms and synaptic plasticity around the ataxic onset of tottering, leaner and tottering leaner double mutation but also Purkinje cell morphology affected by voltage-sensitive calcium channel a1A subunit mutation in totterring mouse. Purkinje cell dendritic spines from proximal dendrites and axonal swellings of Purkine cell were observed frequently in wild type mice. The first apperance point of altered synapse based on semi-quantitative analysis was postnatal 15 days in leaner, postnatal 18 days in totering/leaner double mutation, and 30 days in tottering. These data suggest that altered synapse is associated with ataxia in tottering and leaner mice. Further study is needed to determine whether altered synapse is primary cause of ataxia.
Subject(s)
Adult , Animals , Humans , Mice , Alleles , Ataxia , Axons , Calcium Channels , Cerebellar Ataxia , Cerebellum , Chromosomes, Human, Pair 8 , Dendrites , Dendritic Spines , Epilepsy, Absence , Immunohistochemistry , Mice, Neurologic Mutants , Migraine with Aura , Models, Animal , Movement Disorders , Nervous System Diseases , Neurologic Manifestations , Plastics , SynapsesABSTRACT
Voltage dependent calcium channels (VDCCs) mediate Ca++ influx into cells and are responsible for regulation of a variety of physiological effects. The key functional property of VDCCs are attributed to the calcium-pore forming alpha1 subunit. In this study, distribution pattern of alpha1 subunit (alpha1D, alpha1B, alpha1A, alpha1E) mRNA of VDCCs in developing and adult rat brain was investigated by in situ hybridization histochemistry. In the adult rat brain, each alpha1 subunit mRNA displayed a specific and distinct distribution pattern. alpha1D was highly expressed in the olfactory bulb, dentate gyrus, pituitary gland, pineal gland, hypothalamus, superior colliculus and cerebellum. Relatively low level of alpha1B was expressed throughout the whole brain and strong expression of alpha1A was observed in CA3 area of Ammon's horn, medial geniculate body, inferior colliculus and cerebellum. High level of alpha1E was found in the olfactory bulb, hippocampus, dentate gyrus, medial habenular nucleus and cerebellum. Moreover, alpha1B, alpha1A and alpha1E were expressed only in the nervous system but alpha1D was expressed not only in the nervous system but also in other tissues including liver, heart, lung and skeletal muscle. Generally the expression of alpha1D, alpha1A, and alpha1E subunit was observed from E14 and thereafter the intensity of labeling was gradually increased to P14 and then decreased to the adult level. But the expression of alpha1B subunit was observed from E14 and gradually increased to E20 and P0 and then decresaed. From the differential expressions of VDCC alpha1 subunits in developing and adult rat brain, it is suggested that each type of VDCCs may play a distinct roles in neural and nonneural tissues, and the VDCCs may be related with development of nervous system.
Subject(s)
Adult , Animals , Humans , Rats , Brain , Calcium Channels , Calcium , Cerebellum , Dentate Gyrus , Geniculate Bodies , Habenula , Heart , Hippocampus , Hypothalamus , In Situ Hybridization , Inferior Colliculi , Liver , Lung , Muscle, Skeletal , Nervous System , Olfactory Bulb , Pineal Gland , Pituitary Gland , RNA, Messenger , Superior ColliculiABSTRACT
Chromosome microdissection has become a very powerful approach to generate chromosome band-specific library and painting probes for physical mapping or cytogenetic analysis. Here we have constructed the band-specific painting probe for human chromosome 18p11.1-p11.2 by microdissec-tion and polymerase chain reaction (PCR). We pretreated the microdissected fragments with Topoisomerase I (Topo I) which catalyzes the relaxation of supercoiled DNA, and performed two initial rounds of DNA synthesis with T7 DNA polymerase followed by conventional PCR to enable the reliable preparation of fluorescent in situ hybridization (FISH) probe from a single microdissected chromosome. With this method, it was possible to construct region-specific painting probe for 18p11.1-p11.2. The probe has been used successfully to determine the trisomy 18. Thus this painting probe can be applied to detect the related marker chromosomes in various solid tumors, and the band specific probe pools may be used to analyse the genes.
Subject(s)
Humans , Chromosomes, Human , Cytogenetic Analysis , DNA , DNA Topoisomerases, Type I , DNA, Superhelical , In Situ Hybridization, Fluorescence , Microdissection , Paint , Paintings , Polymerase Chain Reaction , Relaxation , TrisomyABSTRACT
PURPOSE: The analysis of recurring chromosome aberrations has become an integral part of the diagnostic and prognostic workup of many human cancers, and their molecular analyses have facilitated the identification of genes related to the pathogenesis of cancer But the technical limitation of conventional cytogenetic method makes unable us to characterize all recognizable chromosome rearrangements. The generation of chromosome region specific painting probe by PCR amplification of microdissected chromosomal DNA has proven extremely useful in identification of chromosomal derivation for marker chromosomes which are indeterminable by routine chromosome banding analysis. In this study we have constructed and analyzed the band-specific painting probe for unidentified marker chromosomes of renal cell carcinoma cell line, Caki-1 to determine the derivative chromosomes and the painting probes applied to CURC-II to compare the marker chromosomes. MATERIALS AND METHODS: Microdissection was performed on 9q+ and unidentified one of Caki-1, and chromosomal BNAs were amplified by PCR using topoisomerase I and T7 DNA polymerase. Fluorescent in situ hybridization was conducted with biotin labeled PCR products to normal, Caki-1 and CURC- II metaphase chromosomes. RESULTS: With this method, it was possible to construct the band-specific painting probes for markers and the probes hybridized specifically to the dissected regions and derivative chromosomes. The 9q+ and unidentified one were identified as t(9;17)(q34;q21) and t(15;20) respectively. The marker chromosomes - t(9;I 7), der(1 ;17)(ql0;q10), t(15;20), t(?;15), der(1 ;20), t(14;89) were examined same in Caki-1 and CURC-II. CONCLUSIONS: Thus this methodological advance significantly extends the limits of conventional cytogenetic analysis by enabling the analysis of unknown chromosome regions, and these painting probes can be applied to detect the similar marker chromosomes in renal cell carcinoma, and the probe pools for markeys may be used to identify the cancer-relevant genes.
Subject(s)
Humans , Biotin , Carcinoma, Renal Cell , Cell Line , Chromosome Aberrations , Chromosome Banding , Cytogenetic Analysis , Cytogenetics , DNA , DNA Topoisomerases, Type I , In Situ Hybridization, Fluorescence , Metaphase , Microdissection , Paint , Paintings , Polymerase Chain ReactionABSTRACT
Painting probe for HSRs in IMR-32 Neuroblastoma, a pediatric malignant neoplasm of neural crest origin, has a wide range of clinical virulence. The mechanisms contributing to the development of neuroblastomas are largely unclear, but non-random chromosomal changes identified over the past years suggest the involvement of genetic alterations. Amplification of the human N-myc proto-oncogene is frequently seen either in extrachromosomal double minutes or in homogeneously staining regions(HSRs) of aggressively growing neuroblastomas. N-myc maps to chromosome 2 band 24, but HSR have never been observed at this band, suggesting transposition of N-myc during amplification. We have constructed and analyzed the region-specific painting probe for HSR in neuroblastoma IMR-32 to determine the derivative chromosomes. Microdissection was performed on HSR using an inverted microscope with the help of microglass needles and an micromanipulator. We pretreated the microdissected fragments with Topoisomerase I which catalyzes the relaxation of supercoiled DNA, and performed two initial rounds of DNA synthesis with T7 DNA polymerase followed by conventional PCR to enable the reliable preparation of Fluorescent in situ hybridization probe from a single microdissected chromosome. With this method, it was possible to construct the region-specific painting probe for HSR. The probe hybridized specifically to the HSRs of IMR-32, and to 2p24, 2p13 of normal chromosome. Our results suggest there was coamplification of N-myc together with DNA of the chromosome 2p24 and 2p13. Moreover, the fluorescent signals for the amplified chromosomal regions in IMR-32 cells were also easily recognized at a Thus this painting probe can be applied to detect the similar amplification of N-myc in neuroblastoma tissue, and the probe pool for HSR may be used to identify the cancer-relevant genes.