Chromosomal localization of transfected genes by a combination of hot banding and fluorescence in situ hybridization.

We describe the combination of hot banding with fluorescence in situ hybridization as a rapid and efficient method to identify integration sites of transfected DNA sequences in chromosomes. As a test system we used SW480 EJ2, a clonal cell line obtained after transfection of SW480 with pSV2neoEJ, a plasmid containing a point-mutated, c-Ha-RAS oncogene. Nick-translated probes were compared with random primed-labeled probes to evaluate their relative efficiency in fluorescence in situ hybridization. The fluorescence signals were quantified in interphase nuclei by confocal scanning laser microscopy. Nick-translated probes were found to yield better results. Hot banding followed by fluorescence in situ hybridization localized the integration site of pSV2neoEJ in SW480 EJ2 at the site of a translocation on a marker chromosome Xp+. The combination of fluorescence in situ hybridization and hot banding can be used to (a) rapidly and efficiently analyze integration sites in large numbers of transfectants, (b) assess the clonality of transfected cell lines, and (c) localize the site of integration of transfected genes in the recipient genome.

Prognostic significance of CEA immunoreactivity patterns in large bowel carcinoma tissue.

In order to determine the clinical value of CEA detection in large bowel cancer tissue the patterns rather than the intensity of immunoreactivity of CEA reactive antibodies were analyzed in 312 large bowel cancer patients especially in relation to patient survival. CEA immunoreactivity appeared to be distinguishable into a predominantly apical/cytoplasmic and a predominantly membranous pattern. Twenty-four (7.7%) tumours were found to be CEA negative or only focally positive. Two hundred and eighty-three (90.7%) of the carcinomas showed a predominantly apical/cytoplasmic immunoreactivity pattern, whereas 5 (1.6%) of the tumours revealed mostly membranous CEA immunoreactivity. CEA negative or focally positive carcinomas and CEA positive tumours with membranous immunoreactivity were significantly more often observed in the group of poorly differentiated carcinomas (P greater than 0.001), but showed no significant correlation with stage of tumour extension (P = 0.11). Also, these carcinomas demonstrated a more aggressive course in patients compared to CEA positive tumours with an apical/cytoplasmic CEA expression pattern. We, therefore, conclude that determination of the pattern of CEA immunoreactivity in large bowel cancer tissue may enable the detection of subgroups of patients with a poor prognosis.