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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A023
Fundamental questions concerning the early events preceding cancer formation
as well as questions dealing with clonal progression and timing of different
mutations, deletions, etc. remain to be resolved. The melange of cell types
in a typical tumor has long been a problem for researchers analyzing gene
expression in cancer cells. Analysis of RNAs extracted from a whole tumor
does not result in an accurate picture because it originated from a mix of
molecules from all of the different cell types included in the tumor. The
challenge has been to separate distinct cells from the others to analyze the
mRNAs of the selected cells. The ROBOT-MICROBEAM SYSTEM (P.A.L.M. GmbH,
Bernried) was used to procure single cells from a colon adenocarcinoma tissue
slice by Laser Microbeam Microdissection (LMM). With a few laser shots (Laser
Pressure Catapulting; LPC) the laser- isolated specimens were catapulted away
from the object slides directly into the cap of a reaction tube. Reverse
transcription followed by nested PCR amplification of the Ki-ras2 mRNA
allowed to detect point mutations within codon 12 in isolated colon carcinoma
cells by artificial restriction length polymorphism, even from a single
paraffin-embedded archival tumor cell. Point mutations in the Ki-ras2 gene
are common genetic alterations associated with development and progression of
human colon cancer. Our method allows to analyze retrospectively expressed
genes from routine histopathological tissue slices to gain more information
about the molecular defects underlying specific diseases.
IDENTIFICATION OF EXPRESSED GENES IN ARCHIVAL TISSUE BY LASER-MEDIATED
MANIPULATION OF SINGLE CELLS: CLUES TO TUMOR PROGRESSION OR TUMOR
HETEROGENEITY
Lahr G, Schütze K
Laserlabor und Molekularbiologie 1.Med.Abteilung Städt.Krankenhaus
Müchen-Harlaching, München, Germany