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Biomedical subjects

D W Stacey

Publications and source records attributed to D W Stacey.

At least 55 records · Page 3Linked to original sources
PubMed1988

The inhibition of protein synthesis by IgG containing anti-ribosome P autoantibodies from systemic lupus erythematosus patients.

Autoantibodies found in approximately 15% of patients with systemic lupus erythematosus recognize three 60 S ribosomal phosphoproteins P0, P1, and P2. Fab fragments obtained from sera of these patients inhibited globin mRNA translation in an in vitro protein synthesizing system which was reversed by the addition of excess ribosomes. Further studies suggested that these antibodies bind to ribosomes in the intact cell. Thus, when IgG fractions from these sera were microinjected into cultured human fibroblasts [35S]methionine incorporation into cellular proteins was inhibited.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

The ras pathway: a model for the control of proliferation in animal cells.

The studies described above have led to some conclusions as well as some speculations regarding the participation of oncogenes in proliferation. It is important to make a clear distinction between the data described along with the resulting conclusions, and the highly speculative models which have been proposed here to describe these results. On the basis of the results described, several conclusions appear to be indicated. First, it is clear that in normal cells of many types ras proteins are required for proliferation. Second, these proteins are needed for the maintenance of the transformed phenotype induced by some, but not all, oncogenes. Third, the activity of ras proteins is not apparently involved in controlling the activity of phospholipase C or any other identifiable phospholipase. Finally, tumor formation appears to involve the development of a proliferative phenotype which functions independently of ras activity. To explain these data, a model of proliferative control is presented. This model is highly speculative at this time and is based upon the assumption that the "ras pathway" involves the sequential action of cellular genes related to the retroviral oncogenes. The function of this pathway is to pass the proliferative signal generated by an occupied growth factor receptor into the cell. It is clearly needed late in the G-1 phase of the cell cycle, but the "ras pathway" might also be involved in the early events associated with mitogenic stimulation. Due to its central role in the control of proliferation and the fact that tumor cells circumvent its action, ras proteins are postulated to a site of negative proliferative control. After the action of ras and related cellular oncogenes, the proliferative control signal no longer involves the action of a simple linear sequence of protein activities, but might involve multiple, interdependent pathways. This model is primarily of value as a working hypothesis and does not account for many observations central to proliferative control such as the involvement of cell-cell contact, differentiation, and the action of factors which inhibit rather than promote proliferation, such as interferons. The model does summarize the data described and even in the simplest form represents a novel approach to explain proliferative control in terms of the activities of known genes.

Animals↗
PubMed1988

Critical role of cellular ras proteins in proliferative signal transduction.

In the experiments described above, a neutralizing anti-ras antibody was utilized to study the role of ras protein in normal cell proliferation. Initially, it was demonstrated that the antibody was specific for ras protein, and that ras activity was efficiently inhibited. With the neutralizing antibody, it was first shown that ras activity is required for the proliferation of all normal cell types tested. ras activity was required just prior to initiation of S phase. The transforming activity of several retroviral oncogenes was also blocked following anti-ras injection. This included the tyrosine kinase, plasma-membrane-associated proteins, and an oncogene derived from a growth factor. On the other hand, cytoplasmic oncogenes with serine kinase activity were not dependent on ras activity for expression of the transformed phenotype. These observations form the basis of our model for proliferative signal transduction. We propose that the action of either growth factors, their receptor molecules, or related oncogenes initiate an intracellular signal received by ras proteins and then transferred by ras to cytoplasmic serine kinase oncogenes. This signal transduction system directly regulates cellular proliferation. Although further evidence in support of this model is needed, it appears from our studies that the mechanism of signaling between tyrosine kinases and ras proteins might be at the level of phospholipid metabolism. This observation is based on the fact that the mitogenic lipid molecules tested were remarkably dependent on ras activity, even more so than the growth factors or related oncogenes tested. Finally, our work suggests a fundamental distinction between normal and tumor cells. All the normal cell types tested were efficiently inhibited in proliferation by the injected antibody. Tumor cells, on the other hand, were never completely inhibited by the antibody and often were not inhibited at all. The presence of an activated ras oncogene within the tumor assured at least a partial role for ras activity in the proliferation of the mature tumor line. The significance of the observed distinction between normal and tumor cells is not known. The fact that this distinction involves a protein with an apparently critical role in normal proliferation suggests that the observation might be important.

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

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.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

The effects of transcriptional regulatory sequences introduced into a retroviral genome.

Chimeric plasmids in which the herpes thymidine kinase (tk) gene replaced portions of the Rous sarcoma viral genome were used to assess the relationship between viral transcription and that of an exogenous gene located within the viral genome. The entire tk gene and portions of the gene were positioned in both orientations within the viral gag and pol genomic region (which serves as intron for viral env mRNA). Microinjection assays then determined the amount of viral genomic transcription by quantitation of the amount of viral env mRNA produced. Separate injections also assayed for the presence of tk mRNA. Both mRNAs were produced unless the 3' region of the tk gene was present within the viral genome and in the same transcriptional sense. In this case viral env mRNA production was nearly abolished.

Animals↗
PubMed1986

A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene.

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.

Animals↗
PubMed1984

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.

Biological Transport↗
PubMed1984

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.

Animals↗
PubMed1983

Microinjection of vesicular stomatitis virus ribonucleoprotein into animal cells yields infectious virus.

Microinjection of purified transcriptionally active ribonucleoprotein (RNP) complex of vesicular stomatitis virus in vero cells resulted in the production and release of virus. Compared to the release of virus by cells treated with RNP in the presence of DEAE-dextran, the microinjection technique was highly efficient. Microinjection in Xenopus oocytes also resulted in initiation of infection as shown by the synthesis of virus-specific proteins in the cell cytoplasm. It was further observed that RNP stripped of L protein but containing residual NS protein was capable of initiating virus production or protein synthesis when microinjected in vero cells or in oocytes, respectively. Since L and NS proteins are essential for in vitro transcription by RNP, these results suggest that a trace amount of L protein may remain bound to the RNP and a host factor may stimulate residual L activity in vivo.

Animals↗
PubMed1983

Selective ligation of DNA molecules following microinjection.

Avian retroviral DNA is rapidly ligated and transcribed following microinjection into chick cells. To analyze the fidelity of intranuclear ligations, DNA molecules with a variety of protruding ends or flush ends were co-injected into env-deficient Rous sarcoma virus-infected (RSV(-] cells. Two fragments were chosen which could complement the RSV(-) env deficiency upon ligation. This sensitive biological assay indicated that intracellular ligation between molecules with complementary protruding ends or flush ends was relatively efficient. Ligation was not observed with non-complementary termini. Furthermore, when multiple DNA molecules possessing a variety of protruding termini were introduced into the cells, complementary termini were selectively joined. Finally, when viral DNAs which expressed separate genetic markers were co-injected, almost 50% of the virus subsequently released were recombinants, indicating that ligations generally involved multiple molecules.

Avian Sarcoma Viruses↗
PubMed1983

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.

Animals↗
PubMed1982

Effect of intron size on splicing efficiency in retroviral transcripts.

To study the effect of intron size on splicing efficiency we have varied the size of the avian leukosis virus (ALV) env mRNA intron in a cloned ALV genome. This was accomplished by deletion of ALV sequences or insertion of phage lambda DNA. The effect of these modifications on splicing was analyzed by microinjection of the modified clones into RSV(-) chicken cells. Viral env mRNA when transcribed and properly spliced within these cells complemented the RSV(-) env deficiency leading to the production of focus forming units. Using this assay it was shown that deletion of up to 3.7 kb of the 4.68 kb env intron did not inhibit correct splicing nor did insertion of up to 8 kb of phage lambda DNA prevent splicing. Our results indicate that intron size can be varied over a wide range without preventing splicing.

Animals↗
PubMed1982

Participation of subgenomic retroviral mRNAs in recombination.

Envelope glycoprotein (env) mRNA from avian retroviruses was injected into cells transformed by env-deleted Bryan Rous sarcoma virus [RSV(-)]. The genetic deficiency of RSV(-) was complemented, and infectious transforming virus was released for many days after these injections. The long-term activity of the injected env mRNA is believed to be due to reverse transcription of the injected RNA after its incorporation into virus particles. The resulting subgenomic provirus, presumed to be integrated into host DNA, is able to direct the continuous synthesis of additional env mRNA. In some of these cultures, replication-competent viruses appeared many days after injection. The analysis by RNase T1 oligonucleotide fingerprinting showed that the RNA of these virus genomes contained oligonucleotides characteristic of both RSV(-) and the env mRNA injected. In all viruses analyzed the 5' two-thirds and the 3' terminus of the genome were derived from RSV(-) and the env gene from the injected mRNA. Our results thus strongly indicate that these viruses were generated via recombination between RSV(-) and env mRNA. The demonstration of involvement of an mRNA sequence in recombination may be of importance in the divergence of retroviruses and in the mechanism of interaction between retroviruses and host nucleotide sequences.

Alpharetrovirus↗