Dennis W. Stacey

Requirement for c-ras proteins during viral oncogene transformation

Many retroviral oncogenes have been classified into one of several categories based on structure, enzymology and cellular localization1. These genes originated from host cells and are probably derived from genes normally involved in the control of cell proliferation2. The cellular counterparts of three oncogenes have been identified as a growth factor or growth factor receptor3–6; related oncogenes include receptor-like membrane proteins which often express tyrosine kinase activity. These growth factor-related oncogenes are structurally and biochemically distinct from the membrane-associated ras gene family, which bind and hydrolyse GTP7–9. Oncogenes localized primarily in the cytoplasm which probably have serine kinase activity, have also been identified10–12. Although the structure and biochemistry of many oncogenes have been extensively studied, relatively little is known about the functional relationships of oncogene proteins within the cell. An opportunity to study such interaction is provided by the identification of a monoclonal antibody that neutralizes cellular ras proteins when microinjected into cells13. It has been shown previously that the injected antibody inhibits the initiation of S-phase in NIH 3T3 cells14. In the present study we injected this monoclonal antibody into NIH 3T3 cells transformed by a variety of oncogenes. The results show that transformation by three growth factor receptor-like oncogenes depends on c-ras proteins, while transformation by two cytoplasmic oncogenes appears to be independent of c-ras protein.

Cellular ras activity and phospholipid metabolism

Cellular ras activity has been neutralized in 3T3 cells by microinjection of a specific anti-ras monoclonal antibody. The injected antibody efficiently inhibited proliferation in cells treated with a phorbol ester and a calcium ionophore, or with prostaglandin F2 alpha. These treatments were designed to imitate the action of phospholipase C or of phospholipase A2. In addition, the highly efficient mitogenic potential of phosphatidic acid was inhibited by the injected antibody even more efficiently than was serum-induced proliferation. The close reliance of phospholipid-induced mitogenesis upon ras activity suggests that ras proteins are unlikely to function to control the action of a phospholipase.

Microinjection of transforming ras protein induces c-fos expression.

Microinjection of p21ras induced c-fos protein accumulation in three types of 3T3 cells. The induction was rapid and efficient and persisted for many hours. In addition, anti-ras antibody dramatically reduced c-fos accumulation after serum stimulation of injected cells. However, cells which expressed p21ras continuously did not maintain a high level of c-fos expression.

Reversal of transformed phenotype by monoclonal antibodies against Ha-ras p21 proteins

The transforming activities of p21 ras proteins have been determined by micro-injection of these proteins into NIH3T3 cells. In order to facilitate functional studies on the effect of ras proteins on malignant transformation and normal cellular growth, analysis has been made with three monoclonal antibodies (YA6-172, Y13-238 and Y13-259) as originally reported by Furth et al. (J virol 43 (1982) 294). Purified immunoglobulin of Y13-259 has the highest titer of binding to bacterially synthesized p21 ras proteins. Experimental analyses indicate that only Y13-259 antibody will neutralize the transforming activity of the co-injected bacterially synthesized ras protein and the neutralization effect was blocked by co-injection of excess ras protein. In addition, micro-injection of Y13-259 immunoglobulin into transformed NIH3T3 cells (obtained by DNA transfection of NIH3T3 cells with molecularly cloned ras gene) reversed their transformed phenotypes. These results indicate that both bacterially synthesized p21 ras proteins and the natural ras proteins produced in NIH3T3 cells were neutralized by Y13-259 antibody.

Differences in intracellular DNA ligation after microinjection and transfection

An uninterrupted avian sarcoma viral genome terminated by viral long terminal repeat sequences was cloned into a pBR322 plasmid. After introduction into a cultured avian cell, transcription of either the circular plasmid molecule or one linearized within the pBR322 sequences could initiate and terminate at long terminal repeat sequences, yielding full-sized viral RNA. A plasmid DNA molecule linearized by cleavage within the viral pol gene, on the other hand, would have to undergo ligation to yield full-sized viral RNA. Microinjection of each of these three types of DNA into the nuclei of quail cells promoted the release of similar virus titers, indicating that the plasmid DNA cleaved within the viral pol gene had been efficiently and accurately ligated. When plasmid DNA was transfected into quail cells, circular and pBR322-cleaved molecules directed the synthesis of similar virus titers, indicating that they were similarly taken up and utilized by the cells. Compared with these results, plasmid DNA cleaved within the pol gene was reduced in activity over 95% after transfection. This reduction did not result from inefficient ligation but from the generation of mutations (of limited size) during ligation of the transfected molecules. Mutations were not observed after microinjection even into the cytoplasm. Consistent with these findings, transfected DNA termini were found to be joined regardless of their structure, whereas ligation after microinjection required that single-stranded protruding DNA termini be complementary.

Cellular ras activity and tumor cell proliferation

A monoclonal antibody able to specifically neutralize activity of the cellular proto-oncogene ras was microinjected into a variety of normal and tumor cell types in order to determine the requirement for c-ras activity during proliferation. Normal cells were always efficiently inhibited by the antibody, while most tumor cells continued to proliferate without inhibition following the injection. Tumor cells containing a mutant ras gene, however, exhibited an intermediate phenotype and were partially inhibited in proliferation by the injected anti-ras antibody. The mutant c-ras gene appears, therefore, to play a role in proliferation even of the mature tumor cell (although other gene products must also be involved). Mutations in most other tumor cells, however, fall into the class of oncogenes which promote proliferation independently of c-ras activity. In this way tumor cell proliferation is clearly distinguished from that of normal cells studied.

Identification of a sequence likely to be required for avian retroviral packaging

Two assays have been utilized to assess the ability of avian retroviral molecules to be packaged into virus particles. Cloned viral genomic molecules were microinjected into the nuclei of chick cells infected by either a lymphoid leukosis virus or an envelope glycoprotein-deficient sarcoma virus. The titer of focus-forming virus released by injected cells, or the ratio of these to helper virus, is then used to determine packaging efficiency, although biological properties other than packaging might also effect these assays. With either assay, deletions up to 3.0 kbp introduced in the viral gag or pol genes did not affect packaging unless sequences near the SstII restriction site (approximately 150 bp 3 of the splice donor site) were deleted. Deletions differing by 2 bp at the SstII site were found to express radically different packaging efficiencies.

Rapid analysis of small nucleic acid samples by gel electrophoresis

Abstract A rapid and sensitive method for agarose gel electrophoresis is described. By simply miniaturizing a conventional gel electrophoresis apparatus, we have decreased the time necessary for the separation of nucleic acid molecules by a factor of 10. The ability to detect DNA molecules by ethidium bromide fluorescence has simultaneously been increased fivefold. Transfer of DNA from these “minigels” onto nitrocellulose filters followed by hybridization using the procedure of C. M. Southern (1975, J. Mol. Biol. 98 , 503–517) was found to be efficient and rapid. This technique is sufficiently sensitive to detect radioactive quantities of [ 32 P]phosphate-labeled DNA or RNA microinjected into 500 chick embryo fibroblasts.

Microinjection studies of protein transit across the nuclear envelope of human cells

Proteins of various molecular weights were conjugated with rhodamine and microinjected into the cytoplasm or nucleus of HeLa cells. The injected proteins were then localized within the cells at various times thereafter with fluorescence microscopy. Proteins below approx. 60 kD rapidly crossed the HeLa nuclear envelope. Some larger proteins also were able to pass into or out of the nucleus, while others were unable to do so, indicating the selective permeability of the HeLa nuclear envelope to large proteins. The nuclear protein HMG17 accumulated within the nucleus shortly after cytoplasmic microinjection.

Expression of a subgenomic retroviral messenger RNA

When mRNA for avian retroviral envolope glycoprotein (env) was injected into cells transformed by env-deficient Bryan Rous sarcoma virus, the env deficiency of the injected cells was complemented to allow the release of transforming virus for up to 40 hr. When virus spread within the injected culture was allowed to occur, a second phase of transforming virus production by the injected culture began approximately 2 days following injection, continued for many days and often increased to titers well above those seen soon after injection. The requirement for virus spread, along with the genetic properties of virus released long after injection, supported the hypothesis that the second phase of virus production resulted when injected env mRNA was packaged into virus released by injected cells. When this virus infected other cells within the culture the env mRNA was reverse-transcribed to form a subgenomic, proviral-like molecule able to direct the synthesis of env mRNA. Accordingly, it was shown tht neither DNA nor full genomic viral RNA contaminating injected mRNA preparations could account for the results. Evidence that an mRNA can be reverse-transcribed into an active, proviral-like molecule may be of importance in the relationship between retroviruses and their hosts.