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PubMedDate not supplied

Microdissection specimens of connective, chondrous, or Bone Tissue of human osteosarcomas and chondrosarcomas transplanted to athymic nude mice.

Five osteosarcomas and two chondrosarcomas were microdissected to separate tumor compartments of calcified, chondrous, and connective tissue. The compartments were lyophilized separately and transplanted subcutaneously or intramuscularly into nude mice for three, four, and five weeks, respectively. In three of the osteosarcomas and in one of the chrondrosarcomas, calcified tissue induced ectopic bone formation by the host, while cartilaginous tissue induced ectopic bone formation in one of the osteosarcomas and in one of the chondrosarcomas. The tumor-derived connective tissue did not induce osteogenic response in the host tissue. Thus, the ability to develop an osteoinductive response and to produce bone morphogenetic protein seems to be restricted to the population of cells that eventually will, or have, differentiated into bone or cartilage.

Adolescent↗

PubMed1981

Analysis of microdissected cataractous human lenses.

The potential of high-performance liquid chromatography (HPLC) as a technique for studying human cataractogenesis by analyzing minute quantities of lens proteins was demonstrated in a human retinitis pigmentosa (RP) lens. Individual human cortical cataractous lenses were microdissected. A well-defined opaque section was identified, the lens capsule was cut, and the section was removed. A nearby clear lens section was dissected out to serve as a control. Soluble crystallins from the cataractous and adjacent normal sections were subjected separately to gel filtration on a HPLC system. In the RP lens, the protein profiles obtained from an opaque polar-posterior subcapsular section and from nearby clear sections of the same lens were significantly different. The protein profile of an opaque area near the equator of the same RP lens was similar to nearby clear sections. These results show that different opaque zones in a single lens may have unique biochemical and biophysical properties.

Cataract↗

PubMed1980

The effect of clonidine on tubular obstruction in postischemic acute renal failure in the rabbit demonstrated by microradiography and microdissection.

Acute renal failure was produced in vasopression-pretreated rabbits by clamping the left renal pedicle for one hour and removing the opposite kidney. Treatment with clonidine, as antihypertensive drug that blunts the kidney's response to vasopressin, resulted in significantly higher creatinine clearance and urine flow rate in the first 6 hours after unclamping. Clonidine (30 microgram/kg given intravenously 30 minutes before unclamping) also significantly lessened the number of hyaline casts in outer medullary tubules and inner medullary loops of Henle 6 hours after unclamping and reduced the number of abnormal tubular contours in microadiograms produced by infusing barium sulfate into the renal artery at sufficient pressure to rupture glomerular capillaries, causing an escape of contrast material into the tubules. The spaces consistently observed between the ends of barium columns and hyaline casts in microdissection studies and the great lengths of the hyaline casts suggest that hyaline casts obstruct the flow of tubular fluid. Clonidine treatment resulted in fewer, shorter, and thinner hyaline casts. These results indicate that tubular obstruction by hyaline casts plays an important role in early postischemic acute renal failure, and that clonidine's beneficial effect is due in part to a reduction in cast formation.

Acute Kidney Injury↗

PubMedDate not supplied

[Microdissection study of the kidneys of reptiles living under different ecologic conditions].

Microdissectional studies on the kidneys of 24 reptilian species living in different habitats show that the relative length of the distal tubuli is higher in animals adapted to a limited water supply and to fresh-water basins, as compared to animals which have unlimited access to water. No regular changes in the length of proximal, intermediate and connective parts were observed. The increase in the length of the distal tubuli occurs simultaneously among different taxonomic groups of reptiles, indicating the existence of parallelism in evolution of their kidneys.

Adaptation, Physiological↗

PubMed1995

Microdissection based cloning of a translocation breakpoint in a human malignant melanoma.

Chromosome translocations in human malignancies have identified the genomic location of several important growth-regulatory sequences (e.g., cellular oncogenes and suppressor genes). Melanomas are characterized by recurring chromosome alterations, including deletion or translocations of the long arm of chromosome 6 (6q). This report details our efforts to clone the t(1;6)(q21;q14) breakpoint in a malignant melanoma to further our understanding of the biology of these tumors. The strategy utilized combined microdissection of the translocation chromosome, development and characterization of a DNA microclone library, isolation of cosmids and YACs from the breakpoint region, ordering of clones by two-color metaphase/interphase fluorescence in situ hybridization, and finally, identification of a YAC spanning the translocation breakpoint. By analogy to other tumor systems, molecular examination of the chromosome 6 breakpoint may provide insight into the pathobiology of this important neoplasm.

Adult↗

PubMed1995

Chromosome microdissection identifies cryptic sites of DNA sequence amplification in human ovarian carcinoma.

DNA sequence amplification contributes to the multistep process of carcinogenesis, and overexpression of amplified genes has been shown to contribute to the malignant phenotype. Cytogenetic analyses of human tumor cells, including ovarian malignancies, frequently show cytological evidence of DNA amplification in the form of double minutes and homogeneously staining regions. In this report, we have combined the techniques of chromosome microdissection and fluorescence in situ hybridization (P. S. Meltzer et al., Nat. Genet., 1: 24-28, 1992) to identify the composition and chromosomal origin of seven homogeneously staining regions from seven cases of ovarian cancer. Twelve specific chromosome band regions were identified as amplified including 11q, 12p, 16p, 19p, and 19q. These results provide important insights into the organization of amplified sequences within ovarian malignancies and add further to our recognition of regions likely to harbor genes important to the development or progression of ovarian cancer.

Aged↗

PubMed1995

[Chromosome microdissection: applications and prospects].

A new microdissection and microcloning procedure of human banded chromosomes has been described. These micro-techniques allow the construction of band-specific DNA libraries, providing a large number of DNA probes for the identification of gene sequences. These technologies associated with fluorescence in situ hybridization studies are particularly efficient to analyse chromosomal regions involved in human genetic diseases including cancer.

Base Sequence↗

PubMed1995

Direct visualization of the clonal progression of primary cutaneous melanoma: application of tissue microdissection and comparative genomic hybridization.

Human cutaneous malignant melanoma progresses through a series of well defined clinical and histopathological stages. It has been assumed that the neoplastic progression of this disease advances from a common acquired nevus or dysplastic nevus through the primary radial growth phase (RGP), primary vertical growth phase (VGP), and finally to distant metastasis. However, it has never been directly shown that VGP is clonally derived from RGP. Furthermore, it has not been possible previously to conduct a detailed genetic analysis on pure tumor cells from archival material because the lesions are a heterogeneous mixture of normal and neoplastic cells, and the entire specimen must be excised and fixed for clinical diagnosis. This report describes a new approach designed to identify DNA copy number changes in tumor cells from a series of progressive primary stages of cutaneous melanoma archival biopsies. Under direct high-power visualization, cells are procured with a sterile needle from highly specific areas of the tissue section. DNA is extracted from microdissected cells (normal, RGP, and VGP), PCR amplified, fluorescently labeled, and examined by comparative genomic hybridization to determine DNA copy number changes. Data obtained from three representative cases suggest a clonal derivation of VGP cells from RGP. This approach could be useful in identifying the sequence of genetic changes in progressive cutaneous melanoma stages.

Aged↗

PubMed1995

Relationship of cytochrome P450 activity to Clara cell cytotoxicity. IV. Metabolism of naphthalene and naphthalene oxide in microdissected airways from mice, rats, and hamsters.

Parenteral administration of naphthalene produces a dose-dependent and tissue-, species-, and cell-selective lesion of murine Clara cells. The rate and stereoselectivity of naphthalene metabolism by microsomal preparations correlate with tissue and species differences in cytotoxicity. Because earlier studies used microsomes obtained from whole tissue, differences in susceptibility of proximal and distal airways could not be related to differences in the metabolic activation or detoxication of naphthalene. Specific subcompartments of the respiratory system, obtained by microdissection, have been used to study the cytochrome P450-dependent metabolism of naphthalene and the epoxide hydrolase/glutathione transferase-dependent metabolism of naphthalene oxide. The rates of naphthalene metabolism were substantially higher in mouse airways than in comparable airways of hamsters or rats. Rates of metabolism were higher in distal airways than in the trachea of all species studied. Metabolism in mouse airways was highly stereoselective, whereas that in hamster and rat tissues was not. Nonciliated cells at all airway levels in mice were heavily labeled with an antibody to cytochrome P450 2F2; little labeling was observed in any portion of rat and hamster lungs. Postmitochondrial supernatants prepared from mouse and hamster airways metabolized racemic naphthalene oxide to diol and glutathione adducts at substantially higher rates than did comparable preparations from rats. Although glutathione levels varied 2-4-fold at different airway levels in the three species studied, levels at the most susceptible site (mouse distal bronchioles) were as high as or higher than those at other, less susceptible, sites. These studies support the view that the rate and stereoselectivity of naphthalene metabolism to naphthalene 1R,2S-oxide catalyzed by cytochrome P450 2F2 are critical determinants in the species-specific and region-selective cytotoxicity of naphthalene in mice. The lack of major differences in the catalytic activity or enantioselectivity of putative detoxication enzymes (epoxide hydrolase or glutathione transferases) between mouse and hamster tissue, combined with data showing that the differences in the metabolic fate of naphthalene oxide in proximal versus distal airways are not dramatic, suggests that the initial epoxidation of naphthalene is an important factor in site-selective toxicity. These studies support the need to use tissue from defined airway levels for studies on the relationship of biochemical and metabolic factors important in cellular injury by lung toxicants, such as naphthalene, where there are dramatic regional differences in susceptibility to injury within the respiratory system.

Animals↗

PubMed1995

Early detection of Knudson's two-hits in preneoplastic renal cells of the Eker rat model by the laser microdissection procedure.

Hereditary renal cell carcinomas invariably develop by the age of 1 year in Eker rats. At the histological level, renal cell carcinomas develop through multiple stages from early preneoplastic lesions (e.g., phenotypically altered tubules) to adenomas. We previously reported that ionizing radiation induces additional tumors (large adenomas and carcinomas) in a linear dose-response relationship and that loss of heterozygosity (LOH) at chromosome 10, where the predisposing tuberous sclerosis (Tsc2) gene is localized, was found in the renal cell carcinomas which developed from hybrid F1 rats carrying the Eker mutation, indicating that in heterozygotes two events (one inherited, one somatic) are necessary to produce at least large adenomas and carcinomas. This study was designed to examine LOH in the earliest preneoplastic lesions, using a laser microdissection procedure. We could accurately dissect single altered renal tubules out of freeze-dried sections and clearly detected LOH in 4 of 19 altered tubules (21%). This is the first demonstration of LOH in single renal tubules. Our present results support the theory of a second, somatic mutation (second hit) as rate-limiting step of renal carcinogenesis in the Eker rat model of dominantly inherited cancer and the tumor suppressor nature of the Tsc2 gene function.

Animals↗

PubMedDate not supplied

Detection of the von Hippel-Lindau gene deletion in cytologic specimens using microdissection and the polymerase chain reaction.

Previous studies have demonstrated von Hippel-Lindau (VHL) gene abnormalities in renal cell carcinoma. Archival cytologic samples of renal cell carcinomas (RCCs) from two patients were evaluated for allelic loss of the VHL gene. Small clusters of renal carcinoma cells were dissected from cytocentrifuge preparations under direct microscopic visualization followed by single-step DNA extraction and subsequent polymerase chain reaction (PCR). Amplification of DNA from the harvested tumor cells demonstrated a loss of heterozygosity at the VHL gene in five RCC specimens obtained from two patients. The combination of microdissection and PCR enabled DNA studies to be performed on a pure population of tumor cells isolated from heterogeneous samples containing admixed normal cellular elements. Selected genetic studies performed on microscopically targeted cells will help to further the biologic and cytomorphologic characterization of the targeted cell population.

Alleles↗

PubMed1993

Rapid isolation and characterization of amplified DNA by chromosome microdissection: identification of IGF1R amplification in malignant melanoma.

We describe a novel strategy characterizing gene amplification in human neoplasia based on targeting double minutes (dmin) and homogeneously staining regions (hsr) for chromosome microdissection. This strategy allows the rapid generation of an amplification unit microclone library and permits the rapid identification of the chromosomal origin of the amplified sequences following fluorescence in situ hybridization (FISH). This strategy has been applied to an hsr-bearing malignant melanoma cell line, HA-A, which was then demonstrated to encode multiple overexpressed copies of the IGF1R gene. This strategy combines all steps for detection, cloning, mapping and isolation of amplified gene(s) into a single process that is readily applicable to any specimen carrying cytologic evidence of gene amplification.

Base Sequence↗

PubMed1993

Adenylate cyclase activity in microdissected rat liver tissue: periportal to pericentral activity gradient.

Adenylate cyclase activity was measured in microdissected samples from lyophilized cryostat sections of rat liver by means of an improved assay. Livers were obtained from adult Sprague-Dawley rats fasted for 22 hr. Adenylate cyclase activities, basal and those elicited by various agents, were determined in dissected samples from periportal and pericentral regions of the classic liver lobule. In all samples, enzyme activity was strongly stimulated by glucagon, cholera toxin, guanosine-5'-O-(3-thiotriphosphate), sodium fluoride and forskolin. The beta-adrenergic agonist isoproterenol produced very weak, if any, enzyme stimulation. Angiotensin II did not inhibit the activity elicited by lithium chloride and GTP at high concentrations, and pertussis toxin did not enhance the GTP-stimulated activity. We observed a periportal-to-pericentral gradient for basal and agent-stimulated activities.

Adenylyl Cyclases↗

PubMed1993

Application of chromosome microdissection probes for elucidation of BCR-ABL fusion and variant Philadelphia chromosome translocations in chronic myelogenous leukemia.

Fluorescence in situ hybridization (FISH) has become an increasingly important method for assessing chromosome rearrangement. The reciprocal translocation constituting the Philadelphia (Ph) chromosome [t(9;22)(q34;q11)] characterizes more than 90% of patients with chronic myelogenous leukemia (CML). However, in the remaining cases the Ph chromosome (genetically characterized by the fusion of the BCR-ABL genes) is thought to arise through complex translocations that are often not readily apparent using routine chromosome-banding analysis. For this reason we have developed unique band-specific probes for two-color FISH that detect unequivocally the Ph chromosome, and its derivatives. Results of the application of these probes are illustrated by analysis of 11 cases of CML (9 of which contain "variant" translocations). The probes were generated by chromosome microdissection and in vitro amplification of the bands involved in the Ph translocation, leading to an extremely fast and sensitive approach to identify this alteration in leukemic cell populations.

Base Sequence↗

PubMed1996

Analysis of 99 microdissected prostate carcinomas reveals a high frequency of allelic loss on chromosome 8p12-21.

To investigate the possible involvement of a tumor suppressor gene(s) on chromosome 8 in prostatic neoplasms, we performed a comprehensive loss of heterozygosity (LOH) study on 99 tumors from 97 prostate cancer patients. One of the carcinomas was a lymph node metastasis; the other 98 were primary carcinomas. Pure populations of carcinoma cells and normal epithelia were procured by tissue microdissection. Two separate tumor foci were obtained from each of two patients. Microsatellite markers from 25 loci on the short arm and one locus on the long arm of chromosome 8 were used for PCR-based LOH analysis on matched normal and tumor DNA samples. The overall LOH on 8p in this study was 85.9% (85 of 99) of carcinomas. The loss was highest at markers D8S133, D8S136, NEFL, and D8S137 (62,72, 64, and 75%, respectively), which are located at 8p12-21. Seventy-nine of 99 tumors exhibited loss in at least one of these four loci. In contrast, LOH at 8p22 was much lower: 17,18,18, and 19% at D8S549, D8S602, D8S254, and D8S261, respectively, with 25 of 99 tumors showing deletion in one or more of the four loci. All but 5 tumors with deletions in this more distal region had at least one retained locus between the 8p22 deletion and a more proximal region of loss at 8p12-21; 1 tumor had loss at 8p22 but not 8p12-21. This suggests there may be two distinct regions of loss and, therefore, two tumor suppressor genes on this chromosomal arm. The loss on 8p12-21 showed little or no correlation with grade or stage of disease.

Aged↗

PubMed1996

Localization by chromosome microdissection of a recurrent breakpoint region on chromosome 6 in human B-cell lymphoma.

Deletion of the long arm of chromosome 6 (6q) is one of the most common chromosomal alterations in human B-cell lymphomas. Conventional cytogenetic banding analysis and loss-of-heterozygosity (LOH) studies have detected several common regions of deletion ranging across the entire long arm (6q), with no defined recurrent breakpoint yet identified. We describe here a strategy combining chromosome microdissection and fluorescence in situ hybridization (Micro-FISH) to determine a minimal region of deletion along chromosome 6. Seven clinical cases and one cell line of follicular lymphoma containing a t(14;18) and one case of diffuse lymphoma, also with a t(14;18), were used for this study. All nine cases had previously defined abnormalities of chromosome 6 determined by cytogenetic analysis. The results of chromosome dissection were unexpected and in contrast to the suggestion of disparate breakpoints by conventional chromosome banding. Specifically, Micro-FISH analysis provided evidence for a common breakpoint at 6q11 in seven of nine cases. After Micro-FISH analysis, all of the presumed simple deletions of chromosome 6 were carefully reanalyzed and shown to actually represent either nonreciprocal translocations (three cases), interstitial deletions (five cases), or isochromosome (one case). The recurrent proximal breakpoint (6q11) was detected in seven of nine cases, with the minimal region of deletion encompassing 6q11 to 6q21. By analogy to other tumor systems, the identification of recurring breakpoints within 6q11 may suggest that a gene(s) important to the genesis or progression of follicular lymphoma can be localized to this band region.

Chromosome Aberrations↗

PubMed1996

Sequence analysis of rearranged IgVH genes from microdissected human Peyer's patch marginal zone B cells.

The Peyer's patches of the terminal ileum are a source of IgA plasma cells in the intestinal lamina propria of experimental animals. They are also thought to harbour IgA memory cells. However, the microanatomical location of Peyer's patch memory cells, and whether they are also present in man is not known. Human Peyer's patches have a pronounced marginal zone (MGZ) of sIgD-negative B cells. In this study we analysed the sequence of polymerase chain reaction-amplified, rearranged IgVH genes from microdissected MGZ B cells, to determine whether this is a site of B-cell memory in Peyer's patches. We observed that the majority of Peyer's patch MGZ B cells contain heavily mutated IgVH genes and are therefore clearly memory B cells. Sequences of rearranged mutated genes in the MGZ have a pattern of replacement and silent mutations expected of selected products of the affinity maturation process. Related clones, with identical CDR3 but different patterns of mutation, are seen. This suggests that either these memory cells are formed as the germinal centre selection process proceeds, or a memory cell has re-entered the germinal centre for further rounds of mutation. Interestingly, in one patient, the MGZ in the Peyer's patches also contains a proportion of B cells with unmutated IgVH 4.21 genes.

Aged↗

PubMed1997

Detection of hepatitis C virus RNA sequences in hepatocellular carcinoma and its precursors by microdissection polymerase chain reaction.

OBJECTIVE: Chronic hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinoma (HCC), but the mechanism of malignant transformation is unknown. To analyze the association of HCV with HCC, we developed a microdissection technique for the detection by polymerase chain reaction of positive (genomic)- and negative (replicative)-strand HCV RNA in histologically confirmed HCC and the surrounding cirrhotic and macroregenerative nodules. MATERIALS AND METHOD: Five HCCs and one macroregenerative nodule and the surrounding cirrhotic liver tissues of all cases were selected for this study. The method entails extraction of RNA from selected areas of formalin-fixed, hematoxylin-stained histologic sections, followed by strand-specific reverse-transcription double polymerase chain reaction and Southern blotting. RESULTS: Positive- and negative-strand HCV RNA sequences were detected in five of six tumors and the surrounding cirrhotic livers. CONCLUSIONS: These results verify the method of polymerase chain reaction detection of HCV RNA from histologically defined, selected lesions. In addition, the findings suggest that HCV RNA persists and replicates in hepatocytes during malignant transformation.

Aged↗