Chapter 13
Combined in Situ Nick Translation and mRNA FISH
for Simultaneous Detection of Apoptosis and
Expression of Apoptosis-Related Genes in Sections
from Paraffin-Embedded Tissue
NORBERT HECKMANN
Introduction
Simultaneous demonstration of apoptotic cells and apoptosis-related gene
expression on the same tissue section correlated to histomorphological
features, appears to be of great interest.
Apoptosis or programmed cell death is an important factor during the
evolution of various types of cancer. The proto-oncogene bc1-2 is known
to act as an inhibitor of apoptosis. P53 has an apoptosis-inducing function
usually demonstrated at the protein level, but due to the short half-life, an
immunohistochemical detection of wild-type protein is not possible in
most benign tumors.
It is possible to correlate histomorphological architecture of colonic
mucosa with fluorescence staining. Published immunohistochemical studies showed the expression ofbc1-2 in non-tumorous colonic crypts to be
restricted to the proliferative cells of the crypt bases (Watson et al., 1996;
Bosari et al. 1995). During very early stages of colorectal carcinogenesis,
abnormally high levels of Bc1-2 oncoprotein are observed (Mosnier et al.,
1996). They are lost during the evolution from adenoma to carcinoma
(Watson et al., 1996; Kikuchi et al., 1997) and have no prognostic significance in established tumors (Bosari et al., 1995). Apoptosis is increased in
hyperplastic polyps and adenomas but decreased in adenocarcinomas,
paralleled by an increased expression of mutant P53 protein (Kikuchi
et al., 1997). P53 expression is higher in de-novo-carcinomas than in
ex-adenoma carcinomas (Mueller et al., 1996).
Our goal was to develop a technique suitable to detect apoptotic cells
on morphologically quotable histological sections and the expression on
the mRNA-level of apoptosis-inducing and inhibiting genes.
Norbert Heckmann, Saarland University, Institute of Anatomy and Cell Biology,
Homburg, 66421, Germany (phone +49-6841-1626101; fax +49-6841-1626121;
e-mail annhec@med-rz.uni-saarland.de)
PROTOCOL
Combined in Situ Nick Translation and mRNA FISH
for Simultaneous Detection of Apoptosis and
Expression of Apoptosis-Related Genes in Sections
from Paraffin-Embedded Tissue
NORBERT HECKMANN
Introduction
Simultaneous demonstration of apoptotic cells and apoptosis-related gene
expression on the same tissue section correlated to histomorphological
features, appears to be of great interest.
Apoptosis or programmed cell death is an important factor during the
evolution of various types of cancer. The proto-oncogene bc1-2 is known
to act as an inhibitor of apoptosis. P53 has an apoptosis-inducing function
usually demonstrated at the protein level, but due to the short half-life, an
immunohistochemical detection of wild-type protein is not possible in
most benign tumors.
It is possible to correlate histomorphological architecture of colonic
mucosa with fluorescence staining. Published immunohistochemical studies showed the expression ofbc1-2 in non-tumorous colonic crypts to be
restricted to the proliferative cells of the crypt bases (Watson et al., 1996;
Bosari et al. 1995). During very early stages of colorectal carcinogenesis,
abnormally high levels of Bc1-2 oncoprotein are observed (Mosnier et al.,
1996). They are lost during the evolution from adenoma to carcinoma
(Watson et al., 1996; Kikuchi et al., 1997) and have no prognostic significance in established tumors (Bosari et al., 1995). Apoptosis is increased in
hyperplastic polyps and adenomas but decreased in adenocarcinomas,
paralleled by an increased expression of mutant P53 protein (Kikuchi
et al., 1997). P53 expression is higher in de-novo-carcinomas than in
ex-adenoma carcinomas (Mueller et al., 1996).
Our goal was to develop a technique suitable to detect apoptotic cells
on morphologically quotable histological sections and the expression on
the mRNA-level of apoptosis-inducing and inhibiting genes.
Norbert Heckmann, Saarland University, Institute of Anatomy and Cell Biology,
Homburg, 66421, Germany (phone +49-6841-1626101; fax +49-6841-1626121;
e-mail annhec@med-rz.uni-saarland.de)
PROTOCOL