The role of tissue microdissection in cancer research.

Tissue microdissection is a laboratory method that is used to procure specific cells or cell populations from a histology slide under direct microscopic visualization. The recovered cells can be studied with a variety of DNA, messenger RNA, and protein analysis methods, including new high-throughput gene expression and proteomics technologies. This approach is permitting investigators to comprehensivelyexamine the molecular anatomy of cells in tissue sections forthe first time. This article reviews the development and evolution of tissue microdissection techniques, summarizes examples of research studies, and discusses related challenges that the research community must address. Additional information and complete laboratory protocols are available on a website at http://cgap-mf.nih.gov/.

Molecular profiling of tissue samples using laser capture microdissection.

The management of cancer and other genetically based diseases is far from optimal in even our most advanced medical centers. There is still uncertainty regarding how diseases will progress in certain patients, toxicity that must be tolerated with imprecise treatment regimens and significant potential for treatment failure. As our understanding of the complexity of these diseases has increased, it has become clear that we must move toward precisely tailored approaches to treating each individual patient. To that end, a major goal of current medical research is the rapid identification of the specific molecular alterations in each patient's disease. This will enable the design of optimal diagnosis, prognosis and treatment, significantly improving survival. This review describes one important approach to the genetic analysis of disease--molecular profiling--and the tenets and technologies necessary for its success.

H-ras-transformed NRK-52E renal epithelial cells have altered growth, morphology, and cytoskeletal structure that correlates with renal cell carcinoma in vivo.

We studied the effect of the ras oncogene on the growth kinetics, morphology, cytoskeletal structure, and tumorigenicity of the widely used NRK-52E rat kidney epithelial cell line and two H-ras oncogene-transformed cell lines, H/1.2-NRK-52E (H/1.2) and H/6.1-NRK-52E (H/6.1). Population doubling times of NRK-52E, H/1.2, and H/6.1 cells were 28, 26, and 24 h, respectively, with the transformed cells reaching higher saturation densities than the parent cells. NRK-52E cells had typical epithelial morphology with growth in colonies. H/1.2 and H/6.1 cell colonies were more closely packed, highly condensed, and had increased plasma membrane ruffling compared to parent cell colonies. NRK-52E cells showed microfilament, microtubule, and intermediate filament networks typical of epithelial cells, while H/1.2 and H/6.1 cells showed altered cytoskeleton architecture, with decreased stress fibers and increased microtubule and intermediate filament staining at the microtubule organizing center. H/1.2 and H/6.1 cells proliferated in an in vitro soft agar transformation assay, indicating anchorage-independence, and rapidly formed tumors in vivo with characteristics of renal cell carcinoma, including mixed populations of sarcomatoid, granular, and clear cells. H/6.1 cells consistently showed more extensive alterations of growth kinetics, morphology, and cytoskeleton than H/1.2 cells, and formed tumors of a more aggressive phenotype. These data suggest that analysis of renal cell characteristics in vitro may have potential in predicting tumor behavior in vivo, and significantly contribute to the utility of these cell lines as in vitro models for examining renal epithelial cell biology and the role of the ras proto-oncogene in signal transduction involving the cytoskeleton.

Mutagen sensitivity in thymidine kinase- and methyltransferase-deficient human lymphoblastoid cells.

In this study, the effect of thymidine kinase deficiency on the responses of the human lymphoblastoid cell line Raji to methyl methanesulphonate and mitomycin C was investigated. Mutagen sensitivity was measured in terms of cell survival and mutation to hypoxanthine-guanine phosphoribosyltransferase deficiency. Thymidine kinase-deficient Raji cells showed decreased survival and increased mutant frequency relative to wild-type cells following treatments with each of the mutagens used. It is suggested that this may be due to an imbalance in the supply of deoxyribonucleoside triphosphates to the excision repair process. The role of O6-methylguanine-DNA methyltransferase in the repair of DNA damage caused by these mutagens is also discussed.

Evaluation of a new method for determining tracheal tube length in children.

Most paediatric tracheal tubes are marked in centimetres from the tip. In 105 children, nasotracheal tube length was set at the level of the vocal cords such that all 3.0 and 3.5 mm internal diameter tubes were placed with the 3 cm mark at the cords, all 4.0 and 4.5 tubes were set at 4 cm at the cords and all 5.0 and 5.5 tubes were set at 5 cm at the cords. Subsequent chest X ray showed that 79% of the tracheal tubes were in the ideal midtracheal position, one tube was marginally short and 20% of the tubes were marginally long. Neither bronchial intubation nor accidental extubation occurred in any subject. This is an effective method to determine tracheal tube length which may be used for both oral and nasal intubation.

Studies on the mechanism of sulofenur and LY295501 toxicity: effect on the regulation of cytosolic calcium in relation to cytotoxicity in normal and tumorigenic rat kidney cell lines.

Treatment of NRK-52E (normal) and H/1.2-NRK-52E (Harvey-ras transfected NRK-52E) rat kidney epithelial-like cells with two Eli Lilly antitumor compounds, sulofenur and LY295501 (15.6 microM-1000 microM) resulted in concentration- and time-dependent cell killing. Cytosolic Ca2+ became elevated in both cell lines in the presence of extracellular Ca2+ but only minimally in its absence. Both drugs were more toxic to the tumorigenic cells than to the normal cells, but LY295501 was significantly more toxic to both cells. The similarity in toxic response by both cell lines suggests a similar mechanism of toxic action for both drugs. Since LY295501 is highly toxic to tumorigenic cells but has a manageable dose-limiting toxicity it shows excellent potential for use in chemotherapy.

Postoperative nausea and vomiting in paediatric surgical inpatients.

A prospective study of postoperative nausea and vomiting (PONV) was conducted in 415 children presenting for inpatient surgery. The overall incidence of PONV was 18.1%). The highest incidence was in children undergoing ENT procedures and increased with age. Avoidance of intraoperative opioids and the use of local anaesthesia and/or non-steroidal anti-inflammatory drugs reduced the incidence of nausea and vomiting postoperatively.

Effects of thymidine kinase and methyltransferase deficiency on mutagenesis in a human lymphoblastoid cell line.

In this study the effect of thymidine kinase (TK) deficiency on mutagen sensitivity was examined in the human lymphoblastoid cell line Raji. Wild-type and TK-deficient Raji cells were treated with a range of concentrations of ethyl methanesulphonate (EMS) and a range of doses of ultraviolet (UV) light, then examined for mutagen sensitivity as measured by cell survival and mutation to HGPRT deficiency. Dose-dependent responses were observed and TK-deficient cells exhibited decreased survivals and increased mutant frequencies relative to wild-type cells. TK-deficient Raji cells are also deficient in O6-methylguanine-DNA-methyltransferase. This may partially account for their sensitivity to EMS but does not account for the results obtained with UV. It is therefore likely that an additional factor, such as alterations in supply of deoxyribonucleoside triphosphates, may affect the mutagen sensitivity of Raji cells.

Tracheal intubation without neuromuscular block in children.

We have studied 80 healthy children, aged 2-14 yr, undergoing adenotonsillectomy in a double-blind, randomized design. Tracheal intubation facilitated by either suxamethonium 1.5 mg kg-1 or alfentanil 15 micrograms kg-1 was compared after induction of anaesthesia with propofol 3-4 mg kg-1. The quality of tracheal intubation was graded according to the ease of laryngoscopy, position of the vocal cords, coughing, jaw relaxation and movement of limbs. There were no significant differences in the overall assessment of intubating conditions between the two groups, and all children underwent successful tracheal intubation. Fewer patients coughed (P < 0.014) and limb movement was less common (P < 0.007) after tracheal intubation facilitated by suxamethonium. Alfentanil attenuated the haemodynamic responses to tracheal intubation.

Unusual presentation of acute upper airway obstruction caused by an anterior mediastinal mass.

We report an unusual case of acute upper airway obstruction. Inhalation of a foreign body caused choking in a 5-yr-old child, but subsequent investigations revealed a large anterior mediastinal tumour, externally compressing the trachea and the main bronchi. Such a presentation may be deceptive and is important, as general anaesthesia may result in complete airway obstruction with fatal consequences.

Perioperative complications following the use of vancomycin in children: a report of two cases.

We describe two patients in whom rapid administration of vancomycin caused severe hypotension. Possible mechanisms for this effect are discussed, with reference to the role of the anaesthetist as administrator of drugs prescribed by others. Recommendations are made on safe administration of vancomycin with respect to rate of infusion and possible interactions.

Light microscopic immunocytochemical localization of hepatic and intestinal types of fatty acid-binding proteins in rat small intestine.

Monospecific antisera to purified hepatic fatty acid-binding protein (hFABP) and gut fatty acid-binding protein (gFABP) have been used to localize these two proteins in the small intestine of fed rats at the light microscopic level. Pieces of duodenum, jejunum, and ileum were removed from 4-, 10-, 20-, 22-, and 60-day-old Sprague-Dawley rats. Both cryostat and paraffin sections were studied for the presence of hFABP or gFABP by the avidin-biotin immunoperoxidase method. Slides were graded blind for the intensity of staining. Despite the structural and immunological differences between these two proteins, we showed no major differences between their staining patterns or their staining intensity throughout the intestine during postnatal development. The staining for both fatty acid-binding proteins was cytoplasmic. No brush border staining was found. Staining was more intense in the proximal rather than distal intestine, in the villus rather than crypt cells, and in the apex rather than the base of intestinal cells. Shifts in staining patterns, and staining intensity occurring during development may be related to variations in dietary fat intake, rates of cell proliferation, intestinal anatomy, and mechanisms for fat absorption.