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At least 739 records · Page 41Linked to original sources
PubMed1995

Mutation at chromosome 11q23 in human non-familial breast cancer: a microdissection microsatellite analysis.

Allelotypic evaluation of loss of heterozygosity (LOH) has been instrumental in the identification of tumour suppressor genes. Here we report a high incidence of LOH at chromosome 11q23 in non-familial breast cancers with in situ, invasive, and metastatic tumour cells microdissected from archival haematoxylin and eosin (H & E) sections for polymerase chain reaction (PCR)-LOH analysis at polymorphic microsatellite loci. Ninety-four cases of non-familial breast cancer were examined at the D11S29 microsatellite locus on chromosome 11q23. Eighty-three cases (88 per cent) were informative and 35 cases overall (42 per cent) had LOH at this locus, comprising 23 per cent of in situ, 36 per cent of invasive, and 28 per cent of metastatic cancers. The DNA from those cancer cells with LOH was amplified at microsatellite loci D11S554 (11p12-p11.2) and D11S534 (11q13). In 19 of 67 cases overall (28 per cent), LOH occurred solely at 11q23. There was an association between LOH at 11q23 and tumour size > or = 2 cm (P < 0.01) in the overall results and the invasive cancers. The data revealed heterogeneity for LOH at D11S29 in in situ, invasive, and metastatic cells from the same case. In general, however, there was concordance between LOH (or its absence) in in situ and invasive disease. We conclude that the distal part of the long arm of chromosome 11 contains a region involved in breast carcinogenesis and that there is molecular heterogeneity at this chromosomal region in individual breast cancer cells.

Adult↗
PubMed2005

Copy number amplification of 3q26-27 oncogenes in microdissected oral squamous cell carcinoma and oral brushed samples from areca chewers.

Oral squamous cell carcinoma (OSCC) is a worldwide disease. In South Asians, the high prevalence of OSCC is tightly linked to areca chewing. The prognosis for OSCC remains dismal and improvement in early diagnosis may benefit the survival of patients with this disease. Chromosome region 3q26-27 has been shown to carry several oncogenes. By quantitative PCR (Q-PCR), the gene copy numbers of TERC, PI3KCA, ZASC1, and TP63 from microdissected OSCCs have been determined. Copy number amplifications of PI3KCA and ZASC1, a newly identified zinc finger transcription factor, were identified in 30 (65%) and 32 (70%) of 46 primary OSCCs, respectively. Co-amplification of PI3KCA and ZASC1 in 50% of primary OSCCs suggests that they are critical targets of the 3q26.3 amplicon. OSCCs carrying higher levels of PI3KCA and/or ZASC1 copy number amplification were associated with a significantly higher propensity for lymph node metastasis. ZASC1 mRNA expression in OSCC was also associated with lymph node metastasis. In addition, copy number amplification of at least one 3q26-27 oncogene was detected in brush samples from 10 of 22 (45%) patients with oral leukoplakia and 5 of 20 (25%) oral mucosa samples from areca chewers without a visible lesion. These data indicate frequent copy number amplification and overexpression of ZASC1 in OSCC. The findings also suggest the potential use of Q-PCR analysis and brush collection of samples to dissect OSCC risk.

Adult↗
PubMed2001

Maternal insertion of 18q11.2-q12.2 in 18p11.3 of the same chromosome analysed by microdissection and multicolour banding (MCB).

OBJECTIVES: Different aberrations in one chromosome 18 were prenatally detected during each of three different pregnancies of a healthy woman. Routine cytogenetic analysis revealed a morphologically altered maternal chromosome 18 as well. The purpose of the current study was to characterize these cytogenetic changes in detail and thus to clarify the reason for the recurrent appearance of morphologically altered chromosomes 18 in this family. METHODS: As GTG banding did not allow resolution of the kind of aberrations present in these four cases, the following molecular cytogenetic approaches were used: microdissection combined with reverse painting and multicolour banding (MCB) analysis using a chromosome 18 specific probe set. RESULTS: Molecular cytogenetic approaches revealed that fetus 1 had a derivative chromosome del(18)(q11.2q12.2), fetus 2 and the mother had the identical derivative chromosomes ins(18)(pterp11.32::q12.2q11.2::p11.32q11.2::q12.3qter) and fetus 3 had a dup(11.2q12.2). CONCLUSION: Partial monosomy in fetus 1 and partial trisomy in fetus 3 can be explained by crossing over events during maternal meiosis.

Adult↗
PubMed2004

Preparation of human heart for laser microdissection and proteomics.

Proteomics generates information on expressed proteins, and laser microdissection (LMD) is a method that allows enrichment of specific cell types from complex heterogeneous tissue. Together they provide a powerful tool for functional genomic research. Here, we have investigated (i) the effects of fixation and staining on cardiac proteins separated by two-dimensional gel electrophoresis (2-DE) and (ii) feasibility of using LMD to separately prepare myocytes and blood vessels for 2-DE gel analysis. This is the first such study of human heart. The effect of fixation (ethanol or acetone), staining with haematoxylin and eosin in the presence and absence of xylene, and antibody staining was investigated. Proteins were separated by 2-DE and spots detected by silver staining. Quantitative spot analysis showed that contractile proteins were preserved under all conditions, and no significant differences were found when the groups studied were compared with the control group. However, there were differences in the visual quality of the gel patterns. LMD provided enough protein from blood vessels and myocytes to run one large-format (18 x 24 cm) 2-D gel for each subset of cells. Collection of this material took 70 h (approximately 2800 blood vessels and 17,000 myocytes) and resulted in tissue-specific gel patterns for these two structures. In conclusion, the use of haematoxylin and eosin staining without xylene provided the best morphology and did not significantly affect protein spot number.

Cells, Cultured↗
PubMed2006

Proteome analysis of hepatocellular carcinoma by laser capture microdissection.

Hepatocellular carcinoma (HCC) is one of the most frequent visceral neoplasia worldwide and is a multifactorial and multistage pathogenesis that finally leads to the deregulation of cell homeostasis. Laser capture microdissection (LCM) may allow a more ready identification of differences in protein expression in selected cell types or areas of tissue, and microscopic regions as small as 3-5 microm in diameter can be sampled. Here we applied the LCM to the proteomic study of hepatitis B-related HCC and surrounding non-tumor tissues. Proteome alterations were observed using 2-DE and ESI-MS/MS, and alterations in the proteome were examined. Twenty protein spots were selected, of which 11 proteins were significantly altered in the HCC compared with the surrounding non-tumor tissues. Of the proteins that were selected, peroxiredoxin 2, apolipoprotein A-I precursor, 3-hydroxyacyl-CoA dehydrogenase type II, and 14.5-kDa translational inhibitor protein appear to be novel candidates as useful hepatitis B-related HCC markers. This study indicates that LCM is a useful technological method in the proteomic study of cancer tissue. The proteins revealed in this experiment can be used in the future for studies pertaining to hepatocarcinogenesis, or as diagnostic markers and therapeutic targets for HCC associated with hepatitis B virus infection.

Carcinoma, Hepatocellular↗
PubMed1993

Ultrathin-layer zone electrophoresis of lactate dehydrogenase isoenzymes in microdissected liver samples.

A highly sensitive electrophoretic separation of lactate dehydrogenase isoenzymes in 150-microns ultrathin-layer polyacrylamide gels is described. By means of the incorporation of miniature-sized wells into the gel and by executing all the analytical steps under paraffin oil, this technique allows the exact and sharp separation of the lactate dehydrogenase isoenzymes in nanogram-sized microdissected liver tissue samples. With this method the heterogeneous distribution pattern of lactate dehydrogenase isoenzymes in the liver acinus of various mammals could be demonstrated, and a new interpretation of their functional roles is offered.

Animals↗
PubMed1994

Subtraction hybridization cloning of RNA amplified from different cell populations microdissected from cryostat tissue sections.

We describe a generally applicable and easily reproducible method for the cloning of differentially expressed RNA, amplified from small numbers of enriched cell populations, obtained by microdissection from single cryostat sections. The procedure involves homopolymeric A tailing of cDNA synthesized from released RNA using an anchored (NN)T12 primer. Subsequent entire cDNA population polymerase chain reaction amplification was carried out using a biotinylated (X)nT16 primer-adaptor in the presence of biotin-dATP. This biotinylated driver cDNA was then twice hybridized in 50-fold excess to heterologous target cDNA made with nonbiotinylated (Y)nT16 primer; common hybrids and excess driver cDNA were magnetically removed following the addition of streptavidin-coated magnetospheres which bound biotinylated strands, leaving enriched target population sequences. These were then directly amplified through the tails using a primer containing only the target-specific (Y)n sequence. Insertion into a lambda-phage vector was facilitated by means of an EcoR1 site incorporated in the (Y)n primer. Subsequent packaging and transformation into Escherichia coli NM522 resulted in cDNA libraries containing approximately 5 x 10(3)-10(4) pfu. Screening of these primary libraries with cDNA derived from the starting populations yielded a large number of differentially hybridizing clones which are currently under analysis.

Base Sequence↗
PubMed2002

A preservation method that allows recovery of intact RNA from tissues dissected by laser capture microdissection.

We report a novel method for preparing samples for laser capture microdissection. The procedure described here permits extraction of intact RNA while preserving morphology, thus being suitable both for identification of specific cells and for analysis of their gene expression. The method is applicable to both mouse embryos and human tumors and may improve the preparation of cDNA libraries from specific cell types without interfering with histological diagnosis.

Animals↗
PubMed2002

A comparison of hydraulic and laser capture microdissection methods for collection of single B cells, PCR, and sequencing of antibody VDJ.

During the development of B lymphocytes, a series of gene rearrangements assemble the sequences that encode immunoglobulin heavy and light chains (VDJ). Earlier studies of VDJ sequence diversification during expansion of cells in splenic or appendix germinal centers used hydraulic micromanipulation (HM) to collect single B cells for PCR amplification of rearranged antibody heavy and light chain genes. PCR products were directly sequenced without a cloning step. Hydraulic micromanipulation is a very tedious method. Once capability to collect single cells by laser capture microdissection (LCM) was developed, we modified previous tissue staining and fixation methods so that we could collect cells from a given stained tissue section by HM and LCM and directly compare our success rates using these two methods. Cells were alkaline lysed and after two rounds of nested PCR products were recovered and directly sequenced. Because each rearrangement of genomic DNA that occurs to form the immunoglobulin heavy-chain-encoding sequence in developing B cells is unique, this system allowed us to verify our success rate in recovering single lymphocytes from tissue sections and amplifying a single allele. The methods developed have now made LCM an efficient alternative to HM for the collection of single B cells.

Animals↗
PubMed1995

Transcription factors and aldolase B gene expression in microdissected renal proximal tubules and derived cell lines.

Renal expression of the aldolase B isoenzyme and transcription factors previously shown to regulate the aldolase B gene promoter in the liver were analyzed in whole kidney, microdissected tubules, and the two PKSV-PCT and PKSV-PR proximal tubule cell lines derived from transgenic mice. Aldolase B gene expression appeared restricted to the proximal tubule, the site where HNF1 alpha, HNF1 beta, C/EBP alpha, and DBP transcripts were also abundant. Compared to the liver, another organ synthesizing aldolase B, proximal tubules from the kidney were characterized by the absence of HNF3 and the presence of higher ratio of HNF1 beta/HNF1 alpha transcripts. The same features were conserved in both PKSV-PCT and PKSV-PR proximal tubule cell lines. Transactivation experiments in PKSV-PCT cultured cells showed that HNF1 alpha, C/EBP alpha, and DBP behave as transactivators of the 190-bp aldolase B gene promoter, and that HNF1 beta had a low transactivating efficiency. HNF1 beta, as well as HNF3, antagonized the HNF1 alpha-dependent transactivation of the aldolase B promoter. The fact that both HNF1 beta and HNF3 factors play similar negative roles by competitively binding close to or on the HNF1 site could suggest that, in proximal tubule renal cells, HNF1 beta has the same attenuator effect on the aldolase B gene promoter as HNF3 in hepatocytes. Thus, these results indicate that such models of established renal tubule cell lines, which have conserved the same features of parental cells, represent valuable tools for studies of the regulation of genes expressed in proximal tubules of the kidney.

Animals↗
PubMed1993

Microdissection and microcloning of genomic DNA markers from human chromosomal region 11q23.

A human genomic DNA library was constructed by using a microdissection-microcloning procedure with polymerase chain reaction (PCR) techniques on DNA from the chromosome 11q23 region. A total of 450 recombinant pUC clones were isolated from the library. Their insert sizes ranged from 150 to 850 bp with a mean of 320 bp. Fifty pUC clones were randomly selected and analyzed in detail. Southern blot analyses showed that 21 (42%) clones were unique DNA sequences, 20 (40%) clones were repetitive sequences, and 9 (18%) clones had no detectable hybridization. The unique sequences were used further in a secondary screening of a partially digested human genomic DNA library constructed in phage vector, and 4 clones were isolated. The chromosomal locations of these phage clones were confirmed to be in the q23 region of chromosome 11 by fluorescence in situ suppression hybridization. These pUC microclones isolated from the chromosomal region-specific genomic DNA library will be useful in the construction of physical contig maps with yeast artificial chromosome and/or cosmid clones and in the positional cloning of disease-associated genes localized to the q23 region of chromosome 11.

Chromosome Mapping↗
PubMed1994

Isolation of expressed sequences encoded by the human Xq terminal portion using microclone probes generated by laser microdissection.

The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region.

Amino Acid Sequence↗
PubMed1994

Mapping a putative tumor suppressor gene on chromosome 9 bands p21-p22 with microdissection probes.

Deletions of the short arm of chromosome 9 have been observed in a number of malignant cell lines and primary tumor samples using cytogenetic and molecular techniques. These tumors include acute lymphoblastic leukemias, lymphomas, gliomas, melanomas, mesotheliomas, bladder cancer, and lung cancer. The smallest region of overlap (SRO) of these deletions is thought to contain a tumor suppressor gene. A microdissection library was constructed from bands 9p21-p23 to obtain DNA probes that would be useful in further defining the limits of the deletions. Eight single-copy probes were found to be homozygously deleted in at least 1 of the 10 cell lines examined. The mapping of these 8 clones using a panel of cell lines with deletions revealed that 3 probes mapped telomeric to the SRO and 5 clones mapped centromeric to the SRO.

Base Sequence↗
PubMed1996

Localization of human elav-like neuronal protein 1 (Hel-N1) on chromosome 9p21 by chromosome microdissection polymerase chain reaction and fluorescence in situ hybridization.

Hel-N1 is a member of the highly conserved elav family of neuronal genes. It shares considerable sequence homology with HuD, another human member, and both genes are expressed in brain. HuD was recently mapped to chromosome 1p34. Here, we have utilized chromosome microdissection polymerase chain reaction and fluorescence in situ hybridization to map Hel-N1 to chromosome 9p21. The different chromosomal locations of these homologous genes underscore their distinct identities.

Chromosome Mapping↗
PubMed1997

The atomic force microscope as a new microdissecting tool for the generation of genetic probes.

The atomic force microscope (AFM) can be used to visualize and to manipulate biological material with relative case and high resolution. This study was carried out to investigate whether probe sets, specific for subregions of the human genome and useful for the painting of chromosome bands, can be established by PCR amplification of AFM-dissected chromosome regions. Compared to standard microdissection techniques, the AFM can be used with much higher precision for the dissection of the region of interest and subsequent nanoextraction of DNA material. After scanning the area of interest in noncontact mode AFM, chromosome bands were cut by the AFM tip at high force. The genetic material of a single cut attached itself to the tip and was extracted and amplified using degenerate oligonucleotide-primed-PCR. Subsequent to hapten labeling, fluorescence in situ hybridization was performed and chromosome band-specific probes were visualized by standard fluorescence microscopy.

Chromosomes, Human, Pair 2↗
PubMed1994

Progesterone modulation of estrogen receptors in microdissected regions of the rat hypothalamus.

One mechanism whereby progesterone opposes the regulatory actions of estrogen in the brain may include the down-regulation of estrogen receptors. A previous study has shown a small decrease in estrogen binding in the combined preoptic area-hypothalamic area in response to progesterone treatment. To determine if this effect is region specific, gonadectomized/adrenalectomized (GDX/ADX) estrogen-treated female rats were administered a single injection of progesterone (5.0 mg, sc) or a silastic capsule containing crystalline progesterone. Control animals were treated identically but without progesterone exposure. Animals were killed 24 or 72 h after initiating progesterone treatment and estrogen binding was measured in cytosol and cell nuclear extracts of the anterior pituitary, mediobasal hypothalamus (MBH), and preoptic area. A significant effect of progesterone injection on nuclear estrogen binding was observed in the MBH at 24 h. This effect had subsided by 72 h. No effect was observed when progesterone was administered in a continuous fashion. To further examine the regional specificity of the progesterone effect and to determine if males were similarly affected, nuclear-bound estrogen receptors were measured in microdissected brain regions from male and female estrogen-treated GDX/ADX rats treated with 5.0 mg progesterone or vehicle 24 h before sacrifice. A significant decrease in estrogen binding was found in the ventromedial nucleus of the female but not the male. A significant effect of progesterone treatment was found in the periventricular preoptic area of the male.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenalectomy↗
PubMed1996

Chromosome Microdissection-Based Techniques for Genome Analysis

It is now possible to start at a cytogenetically defined position in any eucaryotic genome and proceed toward isolation and identification of candidate genes known to map to that position, taking advantage of the new PCR amplification technology to produce position-specific DNA. The starting material for this very useful exercise is DNA microdissected from standard cytogenetic preparations. Here we describe techniques for acquiring useful samples of such DNA and strategies for the use of chromosome region-specific DNA for gene discovery projects.

Journal Article↗
PubMed1995

Clonal expansion of T cells and HIV genotypes in microdissected splenic white pulps indicates viral replication in situ and infiltration of HIV-specific cytotoxic T lymphocytes.

Human immunodeficiency virus (HIV) replication and T cell proliferation was investigated in situ by a PCR based analysis of individual microdissected splenic white pulps. Founder effects, revealed by an exquisite compartmentalization of HIV genotypes and T cells, indicated the recruitment of latently infected CD4+ T cells through highly localized antigen presentation, rather than the infection of CD4+ T lymphoblasts by blood borne virus or immune complexes. HIV infected white pulps could be infiltrated by HIV specific cytotoxic T lymphocytes, so implicating them in CD4+ T cell destruction in vivo. Together these data describe an iterative and deleterious mechanism of antigen driven T cell recruitment and activation, HIV replication and spread, with consequent destruction of the newly infected cells.

Amino Acid Sequence↗