Tumor specimens from patients with adenocarcinoma of the colon or rectum were examined for the presence of cytomegalovirus (CMV), and specimens of normal mucosa from the same patients were studied in parallel. Frozen sections of 14 specimens were made and the presence of CMV mRNA assayed by in situ hybridisation using 3H-labelled CMV-DNA as a probe. Nine of these sections were also tested for cytomegalovirus antigens by immunofluorescence. No viral nucleic acids or antigens were detected. In addition to these direct approaches, the specimens were disaggregated and 19 were successfully cultured in various media over several months without yielding virus on any occasion. Areas containing epithelial cells were found in some cultures, foci of bipolar cells in others, while, in several, fibroblastic cells predominated. To ensure that any virus-containing cells were not lost by this method, the disaggregated tumour and normal intestinal cells were directly co-cultivated and also fused with human embryo lung cells, which are permissive for cytomegalovirus replication. The resulting cultures were examined over two to three months for the presence of cytomegalovirus, and in no instance was virus found, despite attempted induction by iododeoxyuridine. Two fusion cultures became contaminated with cytomegalovirus, strain AD-169, which was being handled in the laboratory at the same time. The strain was identified by the pattern of viral DNA fragments produced by restriction endonuclease cleavage. Thus the accidental passage of virus in the heterokaryons did not alter its DNA and would further indicate the absence of any cytomegalovirus genomes in the adenocarcinoma cells.
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