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We studied the different potentials of a secreted and a nonsecreted member of the fibroblast growth factor (FGF) family to induce autocrine growth stimulation in human adrenal cortex carcinoma cells (SW-13). These epithelial cells express basic FGF (bFGF) cell surface receptors, and picomolar concentrations of bFGF suffice to induce anchorage-independent growth. The requirement for exogenously added bFGF contrasts with the intracellular storage of biologically active bFGF in SW-13 cells greater than 10,000-fold in excess of the concentration needed to stimulate anchorage independent growth. To study whether the expression of a secreted FGF would alter the growth phenotype of these cells, we transfected them with an expression vector coding for the Kaposi-fgf (K-fgf) oncogene. In contrast to controls, K-fgf-transfected cells secrete significant amounts of biologically active K-fgf protein into the growth media, show up to 50-fold increased colony formation in soft agar, and grow into rapidly progressing, highly vascularized tumors in athymic nude mice. A reversible inhibition of the autocrine growth stimulation in vitro is brought about by the polyanionic compound suramin. We conclude that FGF has to be released from SW-13 cells to function fully as a growth stimulator in vitro and in vivo.

Infection of Friend erythroleukemic (FL) cells by polyomavirus (Py) invariably results in the selection of persistently infected FL-Py cell lines and clones. Anti-Py serum treatment of FL-Py lines and clones leads to the loss of Py genome and consequent cell cure. Conversely, cure has not been obtained in FL-PytsA cell lines (isolated after infection by a Py thermosensitive early mutant) and their derivative clones cultivated for a long time at nonpermissive temperature (39 degrees C), where viral large-T protein is inactive. Rescue of viral particles has always been obtained after shifting cells to 32 degrees C. Integrated viral genomes were detected by blot hybridization in an FL-PytsA clone at 39 degrees C. Long-term observation of FL-Py cell lines and their derivative clones reveals a reciprocal selection mechanism (coevolution) between the viral and the cellular populations, resulting in either a completely virus-free Py-resistant FL cell line (cure) or in a continuously Py-shedding line bearing Py genome variants. Structural analysis of these viral populations has been carried out, and some viral variants have been isolated and characterized. On the basis of the results obtained, the possible mechanisms of Py persistence in FL cells will be discussed.

By transfecting high molecular weight DNA from a Kaposi sarcoma lesion into murine NIH 3T3 cells, we have identified and molecularly cloned a set of human DNA sequences capable of inducing focus formation, growth in agar, and tumorigenicity in these cells. The human DNA sequences present in primary, secondary, and tertiary NIH 3T3 transformants encompass about 32 kilobases (kb) and contain four rearrangements with respect to normal human DNA and a portion of the c-fms protooncogene (FMS in human gene nomenclature). However, the minimal transforming region (6.6 kb) identified in our cloned DNA borders on the c-fms DNA region but does not contain c-fms coding sequences. The fms sequences are also not represented in the two transcripts (approximately equal to 1.2 and 3.5 kb) detected in NIH 3T3 transformants; however, they might provide elements regulating expression. Hybridization to several known oncogene probes and preliminary sequencing data indicate that we have identified a previously unrecognized "activated" oncogene. Since the rearrangements present in our cloned DNA sequences are not detectable in the original Kaposi tumor DNA used for transfection, it is possible that this oncogene was generated during gene transfer.

Friend erythroleukemic cells (FLC) infected by Polyoma (Py) virus were studied at the electron microscope both during their lytic cycle, which occurs within the first 96 hr after infection, and in the surviving stabilized cells 30 days after infection. The results showed that the Polyoma virus and Friend leukemic virus (FLV) coexist and mature together in the same cell. During the lytic cycle, the Py particles were scattered in the nucleoplasm, and sometimes they were also found aggregated into crystals. Instead crystalline aggregates of Py particles were always present in the few surviving virus-carrying cells 30 days after infection. A possible interpretation could be that the crystal aggregation serves to maintain the viruses in a latency that allows the survival of the host cells.

Two mouse neuroblastoma cell lines were analyzed for their permissivity for polyoma virus growth. One (N18) is fully permissive for polyoma replication, the other (41A3) shows limited permissivity and the viral genome persists, without noticeable cell death. Virus persistence does not seem to alter the cells' ability to differentiate in vitro and leads to selection of viral mutants altered in the untranscribed regulatory region of the genome. The mutant types obtained appear to be related to the degree of host cell differentiation. Nucleotide sequence analysis of the restriction fragment covering the regulatory region shows that duplications are present in all mutants, while deletions in the non-duplicated segment are only present in mutants selected from less differentiated cells. These alterations involve both domains of the regulatory region that are considered to be essential for DNA replication and for enhancer activity. Mixed infections with polyoma wild type show that the selected mutants have cis-advantage in replication in neuroblastoma cells and not in 3T6 cells. Mutants carrying the deletion in the non-duplicated segment of the enhancer show a selective advantage in replication over the undeleted one in mixed infection. This advantage is much stronger in neuroblastoma cells in suspension (less-differentiated stage) than in monolayer cells (more-differentiated stage). An interpretation of the overall structure of the regulatory enhancer region, based on the observed differences between the mutants selected at different stages of differentiation in neuroblastoma and previously described mutants selected in undifferentiated cells, is discussed.