Analysis of h-ras, k-ras and N-ras genes for expression, mutation and amplification in laryngeal tumors

Previous work from our laboratory has demonstrated that a clone of Moloney murine sarcoma virus (MoMuSV-349) inoculated intraperitoneally into newborn BALB/c mice induces multiorgan disseminated angiosarcomatous tumors. These tumors develop in two stages: sarcomatous and angiosarcomatous. The present report investigates the relationship between tumor stage development and viral replication within the target cells using the technique of in situ hybridization. A nonradioactive RNA probe (riboprobe) labeled with digoxigenin-linked uridine triphosphate (DIG-UTP) was used to detect the presence of MoMuSV-349 in frozen tissue sections. Infected and control mice were euthanized at various times post-inoculation and tissues were collected for in situ hybridization. The riboprobe hybridized to macrophages, splenic reticular cells, mesothelial cells, megakaryocytes, Kupffer cells and neoplastic (spindled and endothelial) cells in mice infected with MoMuSV-349. The riboprobe failed to hybridize to any tissues in the non-infected mice. Cells hybridizing with the riboprobe were detected as early as 3 days post-inoculation. As the neoplasms progressed from the sarcomatous to angiosarcomatous type there was a shift in cellular target hybridized by the riboprobe from spindled cells to endothelial cells. Positive labeling of endothelial cells during the angiosarcomatous stage of tumor development suggests-that viral replication correlates with cellular proliferation. This demonstrates that in situ hybridization utilizing a non-radioactive riboprobe was useful for a pathogenesis study involving MoMuSV-349.