We have analyzed the transcriptional activity of cellular target sequences for Moloney murine leukemia virus integration in mouse fibroblasts. At least five of the nine random, unselected integration target sequences studied showed direct evidence for transcriptional activity by hybridization to nuclear run-on transcripts prepared from uninfected cells. At least four of the sequences contained multiple recognition sites for several restriction enzymes that cut preferentially in CpG-rich islands, indicating integration into 5' or 3' ends or flanking regions of genes. Assuming that only a minor fraction (<20%) of the genome is transcribed in mammalian cells, we calculated the probability that this association of retroviral integration sites with transcribed sequences is due to chance to be very low (1.6 x 10-2). Thus, our results strongly suggest that transcriptionally active genome regions are preferred targets for retrovirus integration.

In cultures of KNS-62 cells derived from a human lung squamous cell carcinoma, the initial growth arrest in the continuous presence of interferon-gamma (IFN-gamma) turned to cytopathic effects after 2 days of treatment. The remaining viable cells showed grossly distorted morphology, with enlargement and extensions up to 5 cell diameters. The presence of apoptotic cells was shown 3 days after treatment with IFN-gamma. Immunocytochemically, the microtubular structures appeared augmented and highly aggregated. The level of alpha-tubulin-specific mRNA was distinctly increased after administration of IFN-gamma, and the amount of extractable alpha-tubulin protein was reduced. In parallel kinetics experiments, growth arrest by serum depletion or by contact inhibition during confluence resulted in reduced levels of alpha-tubulin-specific mRNA and in slightly elevated alpha-tubulin protein. The IFN-gamma-induced effects suggest interference with assembly or maintenance of the tubulin cable network, presumably associated with cell deformation and cytotoxicity.

The karyotypic and phenotypic stability of cultured rat fibrosarcoma cells was challenged by infection with Moloney murine sarcoma virus (MoMuSV). After transformation, the spindle-like morphology of the parental HH-16 cl.2/1 cells had altered to a rounded phenotype, which was maintained in tumors produced by inoculating transformed cells into congenic animals. In contrast to the parental cells, transformed cells lacked cables of cytokeratins 14-16 and 19 and showed reduction of the mesenchymal marker protein vimentin. Additionally, the morphologically altered cell clones tf-1 to tf-3 had lost growth arrest in the presence of dexamethasone. The DNA of the transformed cells contained between four and six randomly integrated proviral copies. Karyotypic alterations were manifested by reduction of morphologically intact chromosomes in the MoMuSV-transformed cells together with increase of structural aberrations. Three additional markers were identified in the virus-transformed cell clones. Karyotypic instability induced by MoMuSV infection appeared closely related to reduction of the cellular differentiation status, although only cells of clone tf-1 had increased metastatic potential.

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