DNA recombination is sufficient for retroviral transduction.

Oncogenic retroviruses carry coding sequences that are transduced from cellular protooncogenes. Natural transduction involves two nonhomologous recombinations and is thus extremely rare. Since transduction has never been reproduced experimentally, its mechanism has been studied in terms of two hypotheses: (i) the DNA model, which postulates two DNA recombinations, and (ii) the RNA model, which postulates a 5' DNA recombination and a 3' RNA recombination occurring during reverse transcription of viral and protooncogene RNA. Here we use two viral DNA constructs to test the prediction of the DNA model that the 3' DNA recombination is achieved by conventional integration of a retroviral DNA 3' of the chromosomal protooncogene coding region. For the DNA model to be viable, such recombinant viruses must be infectious without the purportedly essential polypurine tract (ppt) that precedes the 3' long terminal repeat (LTR) of all retroviruses. Our constructs consist of a ras coding region from Harvey sarcoma virus which is naturally linked at the 5' end to a retroviral LTR and artificially linked at the 3' end either directly (construct NdN) or by a cellular sequence (construct SU) to the 5' LTR of a retrovirus. Both constructs lack the ppt, and the LTR of NdN even lacks 30 nucleotides at the 5' end. Both constructs proved to be infectious, producing viruses at titers of 10(5) focus-forming units per ml. Sequence analysis proved that both viruses were colinear with input DNAs and that NdN virus lacked a ppt and the 5' 30 nucleotides of the LTR. The results indicate that DNA recombination is sufficient for retroviral transduction and that neither the ppt nor the complete LTR is essential for retrovirus replication. DNA recombination explains the following observations by others that cannot be reconciled with the RNA model: (i) experimental transduction is independent of the packaging efficiency of viral RNA, and (ii) experimental transduction may invert sequences with respect to others, as expected for DNA recombination during transfection.

Different levels of v-Ha-ras p21 expression in primary keratinocytes transformed with Harvey sarcoma virus correlate with benign versus malignant behavior.

Primary mouse keratinocytes transformed with an activated ras oncogene transduced by helper-free Harvey sarcoma virus (HaSV) form predominantly benign tumors. In contrast, keratinocytes transformed with helper-associated HaSV form malignant tumors. We report here that this different tumorigenic behavior correlated with a much higher level of v-Ha-ras p21 protein in cells transformed with the helper-associated virus. The high level of v-ras expression in these cells was due to viral spread beyond the initial infection. The low level of v-ras p21 expression that resulted from single-hit infection with helper-free virus, together with the intrinsic heterogeneity of primary keratinocytes, explains, at least in part, the different tumorigenic behavior of keratinocytes transformed by the two types of viruses.

Enhancement of the proliferation of murine fetal liver erythroid progenitors by infection with Harvey sarcoma virus.

We recently developed a new progenitor assay using murine fetal liver cells that provides a source of pluripotent progenitors, bipotent progenitors, and committed macrophage, megakaryocyte, erythroid, and mast cell progenitors. This clonal cell culture system was used to examine the direct effects of Harvey sarcoma virus on murine hemopoietic progenitors. Very large erythroid colonies containing 100,000 to 200,000 cells were seen in the infected group. Only small erythroid colonies were seen in the uninfected control cultures. The cells in the large erythroid colonies from infected cultures expressed the ras gene as demonstrated by immunofluorescence with a monoclonal antibody to p21, the ras gene product. The infected cells were not immortal since they did not yield secondary colonies upon replating. Sequential observation of individual colonies showed that maturation was not blocked by infection with the virus. The size of other colony types, including granulocyte/macrophage, mast cell, and mixed, was unaffected even though some of these colonies expressed the ras gene. Thus, infection with Harvey sarcoma virus appears to give a growth advantage primarily to committed erythroid progenitors.

Ras p21 onco-protein in the sera of mice carrying an experimentally induced tumor and in human cancer patients.

We have established a model system to detect the presence of ras p21 in the sera of Balb/c mice carrying tumors induced by a mouse cell line transformed with the Harvey murine sarcoma virus in the presence of a helper Friend murine leukemia virus. As determined by ELISA and immunoblot assays, ras p21 in the serum increased with increased tumor growth. Since ras genes have been found to be frequently activated in human tumours, we examined the levels of ras p21 in the sera of a variety of human cancer patients. In only 3 out of 13 cases, representing patients with adenocarcinomas of the stomach receiving chemotherapy, was ras p21 detected at elevated levels, whereas in patients with the following types of cancer no substantial change in serum ras p21 was observed; nine with breast, 5 colon, 5 lung, 5 ovarian and 5 hepatocellular carcinomas.

Rescue of presumptive viral information from human cells by a helper oncovirus.

We have attempted to rescue presumptive human endogenous retrovirus(es) by using a competent animal oncovirus as a helper. Human melanoma cells (line HMB2) were fused, using polyethylene glycol, with mouse NIH-3T3 cells which had been infected and transformed by the Harvey murine leukaemia and sarcoma virus complex (MLV and MSV). The heteropolykaryons obtained were co-cultivated with fresh NIH-3T3 cells; filtered (Millipore 0.22 micron) medium from these was used to infect further NIH-3T3 cells. In these cells after several passages, vesicular stomatitis virus (VSV) pseudotypes could be produced. These were infectious not only for mouse cells (manifesting the helper MLV), but also for human cells (HeLa, HEC human embryo fibroblasts, HMB2); they were not infectious for CCL64 (mink) or for Vero (African green monkey) cells. The presence of such VSV pseudotypes infectious for human cells indicated that a human ecotropic virus [provisionally named rescued human virus (RHV)] had been rescued by the fusion of human melanoma cells with MLV-infected mouse cells. This was supported by the following evidence. The human-specific pseudotype was neutralized by sheep antisera raised to antigens selected by VSV from human tumour cell lines HMB2, T47D and HeLa. These antisera also aggregated NIH cells infected with MLV and RHV. Mouse antisera raised to antigens present in HIH cells infected with MLV and RHV, in contrast to sera raised to NIH cells infected with MLV only, immunoprecipitated an 85,000 mol. wt. protein band from human cells (HEC, HMB2 and HeLa) surface-labelled with 125I.

Infection of immune mast cells by Harvey sarcoma virus: immortalization without loss of requirement for interleukin-3.

Cells from adult mouse spleens were cultured in WEHI-3 cell-conditioned medium, which contains the lymphokine interleukin-3 (IL-3). Under these conditions, cells grow well for 4 to 8 weeks; the cultures contain a variety of cell types for the first 1 to 2 weeks but are subsequently composed largely of immune mast cells. We found that infection of these cultures with Harvey sarcoma virus (HaSV) profoundly enhanced the growth potential of the cells, resulting in the reproducible isolation of long-term cell lines. These HaSV-infected cells appeared to be phenotypically identical to the immune mast cells found in uninfected cultures as determined by biochemical, immunological, and cytological tests. Although the cells expressed protein p21Ha-ras at levels similar to those in HaSV-transformed fibroblasts, they continued to require IL-3 for growth in vitro. Similar IL-3-dependent, long-term mast cell lines were also cultured from the enlarged spleens present in HaSV-infected mice. These results suggest that high-level expression of an activated Ha-ras oncogene enhances growth in these cells, perhaps by stimulating the progression of the cells into S, without affecting differentiation or altering the requirements for normal growth factor.

Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies.

Exposure of neu-oncogene-transformed NIH 3T3 cells to monoclonal antibodies reactive with the neu gene product, p185, results in the rapid and reversible loss of both cell-surface and total cellular p185. Although not directly cytotoxic, monoclonal anti-p185 antibody treatment causes neu-transformed NIH 3T3 cells to revert to a nontransformed phenotype, as determined by anchorage-independent growth. Isotype matched control antibodies of an unrelated specificity do not affect p185 levels or colony formation in soft agar by neu-transformed NIH 3T3 cells. Soft agar colony formation by NIH 3T3 cells transformed by ras oncogenes is not affected by anti-p185 antibody treatment. Anchorage-independent growth of cells from the ethylnitrosourea-induced rat neuroblastoma line in which neu was originally detected by DNA transfection is also inhibited in the presence of anti-p185 monoclonal antibodies. Collectively, these results suggest that p185 is required to maintain transformation induced by the neu oncogene.

Immunocytochemical localization in normal and transformed human cells in tissue culture using a monoclonal antibody to the src protein of the Harvey strain of murine sarcoma virus.

Using a rat monoclonal antibody directed against the p21 src protein of the Harvey strain of Murine Sarcoma Virus (MSV), we have examined the reactivity of human cells in tissue culture using immunofluorescence and electron microscopy. Qualitative results indicated that untransformed mouse and human fibroblastic cells have undetectable amounts of p21; these levels were greatly increased after transformation with Harvey MSV. A group of human tumor cell lines adapted to tissue culture were examined and almost all of the epithelial tumor lines showed significant localization with this antibody. Notable exceptions were two melanoma cell lines which were negative for p21 by immunofluorescence. When normal human epithelial cells derived from esophageal or foreskin epithelium were examined, the antibody showed significant reactivity with subconfluent growing cells. After the normal cells were allowed to become quiescent, the reactivity with this antibody decreased. All of the localization seen by fluorescence was in a distribution consistent with the previously demonstrated location of p21 scr on the inner aspect of the plasma membrane. Electron microscope localization showed labeling for this antigen on the inner surface of the plasma membrane in both transformed mouse cells and in the human tumor cell lines MCF-7 and HTB-2 (RT4). These results suggest that the amounts of p21-like proteins detectable in human epithelial tumor cells do not necessarily reflect their malignant potential, but may be related to their epithelial nature. The loss of detectable localization at quiescence suggests that p21 levels decrease when these epithelial cells stop growing, and raises the possibility that an analog of p21 may be used by these human epithelial cells to regulate cell growth.