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Biomedical subjects

D R Lowy

Publications and source records attributed to D R Lowy.

At least 91 records · Page 5Linked to original sources
PubMed1990

Identification of small clusters of divergent amino acids that mediate the opposing effects of ras and Krev-1.

Krev-1 is an anti-oncogene that was originally identified by its ability to induce morphologic reversion of ras-transformed cells that continue to express the ras gene. The Krev-1-encoded protein is structurally related to Ras proteins. The biological activities of a series of ras-Krev-1 chimeras were studied to test the hypothesis that Krev-1 may directly interfere with a ras function. The ras-specific and Krev-1-specific amino acids immediately surrounding residues 32 to 44, which are identical between the two proteins, determined whether the protein induced cellular transformation or suppressed ras transformation. Because this region in Ras proteins has been implicated in effector function, the results suggest that Krev-1 suppresses ras-induced transformation by interfering with interaction of Ras with its effector.

Amino Acid Sequence↗
PubMed1990

The region of the HPV E7 oncoprotein homologous to adenovirus E1a and Sv40 large T antigen contains separate domains for Rb binding and casein kinase II phosphorylation.

Some genital human papillomavirus (HPV) types, such as 16 and 18, are highly associated with malignant cervical tumors while others, such as HPV 6, are only rarely found in these malignancies. The E7 oncoproteins of HPV 6, 16 and 18 each have a 17 amino acid region with striking homology to adenovirus E1a and SV40 LT. E1a, LT and the E7 oncoprotein of HPV16 all bind the cellular Rb protein in vitro, and for E1a and LT this region of homology contains sequences essential for interaction with Rb. We have now found that in HPV 16 E7 this region (amino acids 21-37) contains two separate biochemical activities, each of which contributes to E7-mediated transformation. Rb binding was localized to the N terminus of this region, while the C terminus was shown to serve as a substrate for casein kinase (CK) II, which phosphorylated serine-31 and serine-32. Replacement of the two serines by non-phosphorylatable amino acids led to a reduction in transforming activity and abolished phosphorylation but did not affect Rb binding. Rb binding and CK II phosphorylation were also examined for the E7 proteins of HPV 6 and HPV 18. HPV 16 and 18 E7 bound similar amounts of Rb, but HPV 6 E7 consistently bound less. Phosphorylation rates also varied, with HPV 18 E7 being 2-fold faster than HPV 16 E7, which in turn was 2-fold faster than HPV 6 E7. We conclude that Rb binding and phosphorylation of E7 by CKII are independent activities which are required for efficient transformation by E7 and that these activities correlate directly with the relative oncogenic potential of these viruses.

Adenovirus Early Proteins↗
PubMed1990

Early regulation of membrane excitability by ras oncogene proteins.

Two electrode voltage clamp conditions were used to study the early effects on ionic membrane channels of the intracellularly injected proto-oncogenic form of c-Ha-ras (c-ras) and its oncogenic counterpart v-Ha-ras (v-ras). These experiments were conducted on isolated somata of identified fully differentiated neurons of the sea snail Hermissenda. 20 min after c-ras, and 10 min after v-ras intracellular injections into type B medial photoreceptors of Hermissenda, the peak amplitude of two outward potassium currents (IA and IC), across the isolated Type B soma membrane begin to decrease. These two currents have been previously isolated by differences in activation and inactivation kinetics and their response to pharmacological blockers. c- or v-ras injections did not have any effect on a voltage-dependent inward calcium current. Reduction of IA preceded that of IC. Current reductions due to c-ras, but not to v-ras injection reversed spontaneously after 40 min. The voltage dependence of the steady state inactivation of IA shifted toward more negative potentials with ras injections. Ras-mediated cell transformations therefore, could involve, perhaps as initial events, prolonged modification of membrane currents.

Animals↗
PubMed1990

Transfected human beta-polymerase promoter contains a ras-responsive element.

beta-Polymerase is a vertebrate cellular DNA polymerase involved in gap-filling synthesis during some types of genomic DNA repair. We report that a cloned human beta-polymerase promoter in a transient expression assay is activated by p21v-rasH expression in NIH 3T3 cells. A decanucleotide palindromic element, GTGACGTCAC, at positions -49 to -40 in the promoter is required for this ras-mediated stimulation.

Animals↗
PubMed1990

Selective potentiation of c-fps/fes transforming activity by a phosphatase inhibitor.

Human c-fps/fes when expressed at sufficiently high levels in NIH 3T3 cells can induce cellular transformation. To probe the mechanism of action of the c-fps/fes product NCP92, we have examined the biological and biochemical consequences of stabilizing phosphotyrosine in cells that overexpress NCP92. In this study we report that when cells expressing c-fps/fes are incubated with low concentrations of sodium vanadate, a tyrosine phosphatase inhibitor, the transforming activity of c-fps/fes can be potentiated by nearly two orders of magnitude. This effect was associated with a threefold increase in the level of phosphotyrosine in NCP92 and its major cellular substrates. Unlike c-src, NCP92 had a single tyrosine phosphorylation site, and vanadate treatment induced its phosphorylation in vivo. This was found to have a positive effect on NCP92 kinase specific activity, but at the low concentrations of vanadate that were used, this effect was very small. The results are consistent with the hypothesis that potentiation was a consequence of the stabilization of phosphotyrosine in critical targets of transformation of NCP92, rather than from a direct effect of vanadate on NCP92 kinase. The potentiating effect of vanadate was relatively specific for c-fps/fes; this reagent did not affect the high transforming activity of gag-v-fps/fes, nor did it enhance the less active c-ras, c-src, or EGF receptor genes, although the latter two genes encode tyrosine kinases. The specificity of the biological response of c-fps/fes to the stabilization of phosphotyrosine suggests that this molecule has a distinct mode of regulation and mechanism of action.

Animals↗
PubMed1989

HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes.

The human papillomavirus types (HPVs) most often associated with cancer of the cervix, such as HPV16, have been reported previously to immortalize normal human foreskin keratinocytes in vitro, while the types that are primarily associated with benign cervical lesions failed to do so. In this study we have determined the HPV16 genes that are responsible for the immortalizing activity of the viral genome. Transfection with a plasmid in which E6 and E7 were the only intact open reading frames (ORFs) induced an indefinite life-span in the keratinocytes with an efficiency similar to that of the entire early region of the viral DNA. Mutants in the E6E7 clone with inactivating lesions in E6 or E7 failed to induce immortalization. When transfected alone, E7 could induce hyperproliferation, but these cells eventually senesced. By itself, E6 exhibited no activity, Co-transfection of a plasmid with an intact E6 ORF and a second plasmid with an intact E7 ORF generated keratinocyte lines with indefinite growth potential. The E6 and E7 proteins were detected in the lines induced by the E6E7 DNA and by co-transfection of the E6 and E7 plasmids. Therefore, we conclude that HPV16 E6 and E7 cooperative to immortalize human keratinocytes in vitro. Changes in cellular gene expression are probably also required for immortalization since all of the keratinocyte lines examined were aneuploid. Serum and calcium resistant sublines were isolated from the E6E7 induced lines, indicating that other HPV genes do not play an obligatory role in the generation of resistance to differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗
PubMed1989

The bovine papillomavirus E5 transforming protein can stimulate the transforming activity of EGF and CSF-1 receptors.

The bovine papillomavirus E5 transforming gene encodes a 44 amino acid protein product that is localized to cytoplasmic membranes, including the plasma membrane. We now report that E5 can cooperate with human EGF receptors and with human CSF-1 receptors to induce cellular transformation of NIH 3T3 cells. Cooperation occurred in the absence of receptor stimulation by ligand, and it was further augmented by treatment with ligand. Cooperation was not seen between E5 and either c-fes or c-src. The cooperation between E5 and high levels of EGF receptors was associated with inhibition of receptor degradation and persistence of activated receptors on the cell surface. We conclude that E5 may enhance the receptor activity via inhibition of receptor down-modulation.

Animals↗
PubMed1989

Retroviruses expressing different levels of the normal epidermal growth factor receptor: biological properties and new bioassay.

Two retroviral DNAs that encode the normal human epidermal growth factor (EGF) receptor hEGFR have been generated by inserting a hEGFR cDNA into two different retroviral vectors. One DNA (pCO11-EGFR-neo) also contained a linked selectable marker gene (neoR). The other (pCO12-EGFR) only expresses hEGFR. When introduced into NIH3T3 cells, the two DNAs and the viruses derived from them induced a fully transformed phenotype, including focal transformation and growth in agar or low serum, but transformation depended entirely upon EGF being present in the growth medium. Compared with pCO11-EGFR-neo, pCO12-EGFR induced EGF-dependent transformation 2-5 times more efficiently and expressed higher numbers of receptors (4 x 10(5) vs. 1 x 10(5) EGF receptors per cell). The results indicate that transforming potential is directly related to the number of EGF receptors. In defined, serum-free medium that contained only very low concentrations of insulin (0.6 microgram/ml) and transferrin (0.6 micrograms/ml), hEGFR-virus infected cells were able to grow with EGF as the only growth factor. Moreover, daily incubation of the cells with EGF for only 30 min was sufficient to induce growth. NR6 cells, which lack endogenous EGF receptors, were transformed as efficiently as NIH3T3 cells by the hEGFR virus. The dose-dependent growth response to EGF of infected NR6 cells grown in serum-free medium can be used as a highly sensitive bioassay for the quantitative assessment of EGF and transforming growth factor type alpha (TGF alpha). This bioassay is at least as sensitive as previously reported radioimmunoassays and can measure a much wider concentration range (10 pg-100 ng/ml). Uninfected NR6 cells or NR6 cells infected by helper virus alone can be used as controls for the EGF specificity of growth stimulation.

Biological Assay↗
PubMed1989

Infectious papillomavirus in the vapor of warts treated with carbon dioxide laser or electrocoagulation: detection and protection.

Papillomavirus DNA has been reported recently in the vapor (smoke plume) derived from warts treated with carbon dioxide laser; this raises concerns for operator safety. We therefore have studied a group of human and bovine warts to define further the potential risk of wart therapy and to test whether a surgical mask could reduce exposure. Half of each wart was treated with carbon dioxide laser and the other half with electrocoagulation. The vapor produced by each form of therapy was collected with a dry filter vacuum apparatus and analyzed for the presence of papillomavirus. Vapor from human plantar warts was analyzed for the presence of human papillomavirus DNA, because there is no infectivity assay for human papillomavirus. Of plantar warts treated, five of eight laser-derived vapors and four of seven electrocoagulation-derived vapors were positive for human papillomavirus DNA. Greater amounts of papillomavirus DNA were usually recovered in the laser vapor than in the electrocoagulation vapor from the same wart. Bioassay readily detected infectious bovine papillomavirus in the vapor from bovine warts treated with either modality; more virus was present in laser-derived material. A surgical mask was found capable of removing virtually all laser- or electrocoagulation-derived virus, strongly suggesting that such masks can protect operators from potential inhalation exposure to papillomavirus.

Air Microbiology↗
PubMed1989

Successive steps in the process of immortalization identified by transfer of separate bovine papillomavirus genes into rat fibroblasts.

Transfer of neor and bovine papillomavirus type 1 (BPV1) DNA into rat embryo fibroblasts led to colony formation in G418-containing medium, with no detectable background in controls with neor DNA alone. More than 50% of the drug-resistant clones could be further propagated in culture. The genetic functions of BPV1 involved in colony formation and in long-term immortalization were investigated by both translation termination mutations in the full-length genome, which inactivate individual open reading frames, and constructs in which these open reading frames were separately expressed under control of long terminal repeat promoter enhancers. Expression of either open reading frame E2 or E5 was sufficient for formation of a drug-resistant colony, but long-term growth in culture required that of E6. No significant cooperative effect was observed upon cotransfection of BPV1 and ras oncogene DNAs. Expression of the early region of the human papillomavirus type 16 also led to immortalization of rat embryo fibroblast cells in the same assay, and, unlike what was previously reported in baby rat kidney cells, it required neither activation by a heterologous promoter, nor a cooperating ras oncogene.

Animals↗
PubMed1989

A highly efficient retroviral vector allows detection of the transforming activity of the human c-fps/fes proto-oncogene.

We have constructed an efficient new retroviral vector containing strong promoting elements derived from the Friend murine leukemia virus (F-MuLV) long terminal repeat (LTR) and have used the vector to demonstrate that overexpression of human c-fps/fes can transform established mouse cells. When a c-fps/fes cDNA was cloned into the vector, this viral DNA and the recovered virus induced very high levels of the c-fps/fes product NCP92 and tumorigenic transformation of NIH 3T3 cells. Compared with an isogenic vector under control of a Moloney MuLV-derived LTR, the vector driven by the F-MuLV LTR induced 3- to 10-times-higher levels of expression of c-fps/fes, a higher level of phosphotyrosine in cellular proteins, and a virus whose transforming activity was 2 orders of magnitude greater. We conclude (i) that normal c-fps/fes can induce morphologic transformation and that its transforming activity is a function of the level of expression of NCP92 and (ii) that the vector based on the F-MuLV LTR is more efficient than the vector driven by a Moloney MuLV LTR in inducing high levels of expression and measurable biological activity.

Animals↗
PubMed1989

Harvey murine sarcoma virus: influences of coding and noncoding sequences on cell transformation in vitro and oncogenicity in vivo.

The rat-derived Harvey murine sarcoma virus (Ha-MuSV) contains a transduced ras oncogene activated by two missense mutations and flanked by rat retroviruslike VL30 sequences. Ha-MuSV induces focal transformation of mouse NIH 3T3 cells in vitro and tumors (fibrosarcomas and splenic erythroleukemias) in newborn mice. We have used these two assays to study the contribution of coding and noncoding viral sequences to the biological activity of Ha-MuSV. A good correlation was found between the in vitro and in vivo assays. In several different isogenic Ha-MuSV variants, those with a rasH gene that had one or both of the Ha-MuSV missense mutations were much more active biologically than the corresponding proto-oncogene. A Ha-MuSV variant that encoded the proto-oncogene protein induced lymphoid leukemias (with thymomas), with a relatively long latent period, rather than the fibrosarcomas and erythroleukemias characteristic of Ha-MuSV with one or both missense mutations. A VL30-derived segment with enhancer activity was identified downstream from v-rasH. A mutant Ha-MuSV from which this 3' noncoding segment was deleted expressed lower levels of the wild-type viral protein, displayed impaired transforming activity in vitro, and induced lymphoid leukemias (with thymomas). 5' noncoding rat c-rasH sequences were found to increase the biological activity of the virus when substituted for the corresponding segment of v-rasH. We conclude that (i) the biological activity of Ha-MuSV can be influence significantly by noncoding sequences located outside the long terminal repeat as well as by coding sequences, (ii) VL30 sequences positively regulate the expression of v-rasH, (iii) relatively low biological levels of ras, whether resulting from low-level expression of wild type v-rasH or high-levels of ras proto-oncogene protein, induce a type of tumor that differs from tumors induced by high biological levels of ras, and (iv) the in vivo pathogenicity of the Ha-MuSV variants correlated with their transforming activity on NIH 3T3 cells.

Animals↗
PubMed1989

Papillomavirus polypeptides E6 and E7 are zinc-binding proteins.

Papillomavirus proteins E6 and E7 have Cys-X-X-Cys repeats which have been suggested to mediate zinc binding. We have developed a modification of an assay that detects zinc binding to proteins immobilized on filters. Using well-characterized metalloproteins, we show that, under reducing conditions, this assay distinguishes proteins that coordinate zinc through cysteine residues from those that bind the metal through other amino acids. Under these conditions, E6 and E7 polypeptides of human papillomavirus type 18 and bovine papillomavirus type 1 exhibited high-affinity zinc binding. Our results suggest that E6 and E7 are metalloproteins and may coordinate the metal ions through cysteine residues.

Carrier Proteins↗
PubMed1989

Identification of bovine papillomavirus E1 mutants with increased transforming and transcriptional activity.

The E1 open reading frame of bovine papillomavirus type 1 (BPV) has been shown previously to encode trans-acting functions, M and R, that are involved in extrachromosomal replication of the viral genome. We have determined that several E1 mutants mapping in both the M and R regions and a single mutant of the upstream regulatory region have a higher transforming activity on mouse C127 cells than the wild-type genome does. A representative mutant in M, a mutant in R, and the upstream regulatory region mutant were complemented in trans by the wild-type genome, but the two E1 mutants did not complement each other, suggesting that they affect the same inhibitory function. A long terminal repeat-activated clone constructed to express the intact E1 open reading frame reversed the high-transformation phenotype of the mutants. In contrast to the high-copy-number autonomous replication of the wild-type genome, the genomes of the E1 mutants were, as previously described for other E1 mutants, integrated at lower copy numbers in the transformed cells. Relative to the viral genome copy number, both the E1 M and R mutant transformed cells contained an average of 10-fold more BPV-specific transcripts than did the wild-type transformed cells. Cycloheximide treatment of the cells transformed by the E1 mutants did not lead to the rapid 10-fold increase in the accumulation of viral transcripts observed with the wild-type genome. These results suggest either that integration of the BPV genome makes it unresponsive to a labile repressor or that an E1 gene product, containing both M and R sequences, is a repressor of BPV transcription.

Base Sequence↗
PubMed1989

Mutational analysis of bovine papillomavirus E6 gene.

The bovine papillomavirus E6 gene can independently transform mouse C127 cells. To characterize E6 in greater detail, we created 16 site-directed mutations in E6, including substitution mutations in the cysteine codons of the four Cys-X-X-Cys motifs that are conserved in all papillomavirus E6 proteins. Proteins mutated in six of the seven cysteines tested, as well as those lacking the nonconserved C-terminus, were stable in transfected cells but were unable to induce morphological transformation, indicating that these amino acids play an important role in the function of E6.

Amino Acid Sequence↗
PubMed1989

Functional heterogeneity of proto-oncogene tyrosine kinases: the C terminus of the human epidermal growth factor receptor facilitates cell proliferation.

Previous reports have indicated that the C termini of the membrane-associated tyrosine kinases encoded by c-src and c-fms proto-oncogenes have a negative effect on their biological activity and that this effect is mediated by their C-terminal tyrosine residue. To determine whether this was true for the human epidermal growth factor (EGF) receptor, which is also a membrane-associated tyrosine kinase proto-oncogene, we have constructed two premature termination mutants, dc19 and dc63, that delete the C-terminal 19 and 63 amino acids, respectively, from the human full-length receptor (hEGFR). The smaller deletion removes the C-terminal tyrosine residue, while the larger deletion removes the two most C-terminal tyrosines; similar deletions are found in v-erbB. As previously shown for the gene encoding the full-length EGF receptor, the two C-terminal mutants induced EGF-dependent focal transformation and anchorage-independent growth of NIH 3T3 cells. However, both dc19 and dc63 were quantitatively less efficient than the gene encoding the full-length receptor, with dc63 being less active than dc19. Although the C-terminal mutants displayed lower biological activity than the gene encoding the full-length receptor, the mutant receptors were found to be similar in several respects to the full-length receptor. These parameters included receptor localization, stability in the absence of EGF, receptor half-life in the presence of EGF, EGF binding, extent of EGF-dependent autophosphorylation in vitro, and EGF-dependent phosphorylation of an exogenous substrate in vitro. Therefore, the C-terminal 63 amino acids of the human receptor have no detectable influence on EGF-dependent early events. We conclude that in contrast

Animals↗