Search PubMed⌕ Search

Biomedical subjects

D DiMaio

Publications and source records attributed to D DiMaio.

At least 37 records · Page 2Linked to original sources
PubMed1997

Identification of amino acids in the transmembrane and juxtamembrane domains of the platelet-derived growth factor receptor required for productive interaction with the bovine papillomavirus E5 protein.

The bovine papillomavirus E5 protein forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in receptor activation and cell transformation. Amino acids in both the putative transmembrane domain and extracytoplasmic carboxyl-terminal domain of the E5 protein appear important for PDGF receptor binding and activation. Previous analysis indicated that the transmembrane domain of the receptor was also required for complex formation and receptor activation. Here we analyzed receptor chimeras and point mutants to identify specific amino acids in the PDGF beta receptor required for productive interaction with the E5 protein. These receptor mutants were analyzed in murine Ba/F3 cells, which do not express endogenous receptor. Our results confirmed the importance of the transmembrane domain of the receptor for complex formation, receptor tyrosine phosphorylation, and mitogenic signaling in response to the E5 protein and established that the threonine residue in this domain is required for these activities. In addition, a positive charge in the extracellular juxtamembrane domain of the receptor was required for E5 interaction and signaling, whereas replacement of the wild-type lysine with either a neutral or acidic amino acid inhibited E5-induced receptor activation and transformation. All of the receptor mutants defective for activation by the E5 protein responded to acute treatment with PDGF and to stable expression of v-Sis, a form of PDGF. The required juxtamembrane lysine and transmembrane threonine are predicted to align precisely on the same face of an alpha helix packed in a left-handed coiled-coil geometry. These results establish that the E5 protein and v-Sis recognize distinct binding sites on the PDGF beta receptor and further clarify the nature of the interaction between the viral transforming protein and its cellular target.

Amino Acid Sequence↗
PubMed1996

Activation of the endogenous p53 growth inhibitory pathway in HeLa cervical carcinoma cells by expression of the bovine papillomavirus E2 gene.

We previously showed that expression of the bovine papillomavirus (BPV) E2 gene results in a dramatic inhibition of the proliferation of several human cervical carcinoma cell lines, including HeLa cells which contain human papillomavirus (HPV) type 18 DNA. We have assessed the status of endogenous G1 cell cycle regulatory proteins, including the tumor suppressor proteins, p53 and p105Rb, in order to investigate growth regulatory pathways in HeLa cells following E2 expression. The p53 tumor suppressor protein is stabilized following the introduction of the E2 gene into HeLa cells. This results in the induction of the p53-responsive gene encoding the cyclin dependent kinase (cdk) inhibitor, p21/WAF1, complex formation between p21/WAF1 and cdk2 and reduction of in vitro cdk2/cyclin E kinase activity. The reduced cdk kinase activity is accompanied by the accumulation of the growth inhibitory hypophosphorylated form of the tumor suppressor protein, p105Rb. The level of the p105Rb-regulated transcription factor, E2F1, is reduced, as is transcription of a variety of E2F1-regulated genes, including B-myb. Thus, the p53 growth inhibitory pathway has evidently not accumulated mutations in HeLa cells but rather appears intact. However, this pathway remains dormant, until it is mobilized by appropriate manipulations, such as the expression of the BPV E2 protein.

Bovine papillomavirus 1↗
PubMed1995

An intact PDGF signaling pathway is required for efficient growth transformation of mouse C127 cells by the bovine papillomavirus E5 protein.

The bovine papillomavirus type 1 (BPV) E5 protein is a 44 amino acid, membrane-associated protein that induces growth transformation of cultured rodent and bovine fibroblasts. In transformed fibroblasts, the BPV E5 protein activates the endogenous platelet-derived growth factor (PDGF) beta receptor, and the introduction of the PDGF beta receptor gene into heterologous cell types normally lacking PDGF beta receptor expression permits transformation by the E5 protein. However, neither the endogenous PDGF beta receptor nor its signaling pathway has been shown to be required for efficient growth transformation of fibroblasts by the BPV E5 gene. Here we have tested whether the endogenous PDGF beta receptor serves as a target for the BPV E5 protein in mouse C127 fibroblasts. We isolated variant C127 cell lines that displayed reduced DNA synthesis in response to PDGF but a normal response to other mitogens, suggesting that they harbor specific defects in the PDGF signaling pathway. The variant lines also exhibited a specific reduction in the level of DNA synthesis induced by the acute expression of the BPV E5 gene, and a variant cell line containing a reduced level of PDGF beta receptor also displayed reduced stable growth transformation by the BPV E5 and v-sis oncogenes. These results provide genetic support for the model that the PDGF beta receptor signaling pathway is required for efficient growth transformation of C127 cells by the BPV E5 gene.

Animals↗
PubMed1995

Mutational analysis of the interaction between the bovine papillomavirus E5 transforming protein and the endogenous beta receptor for platelet-derived growth factor in mouse C127 cells.

The bovine papillomavirus E5 protein is a 44-amino-acid membrane-associated protein that forms a stable complex with the endogenous platelet-derived growth factor (PDGF) beta receptor in rodent and bovine fibroblasts, resulting in sustained receptor activation and cell transformation. We report here that high-level expression of the E5 protein caused a reduction in the level of the mature form of the PDGF beta receptor in acutely and stably transformed mouse C127 cells. To explore in more detail the interaction of the E5 protein and the PDGF beta receptor, we tested the abilities of various E5 point mutants to bind the PDGF receptor, to induce PDGF receptor down-regulation and tyrosine phosphorylation, and to transform cells. A transformation-competent mutant, like the wild-type E5 protein, bound the receptor and induced receptor tyrosine phosphorylation and down-regulation. Transformation-defective E5 proteins either failed to interact with the endogenous PDGF beta receptor in mouse fibroblasts or underwent an aberrant interaction with the receptor. Mutation of glutamine at position 17, aspartic acid at position 33, or both carboxyl-terminal cysteine residues required for E5 homodimerization interfered with stable complex formation with the PDGF receptor, tyrosine phosphorylation and down-regulation of the receptor, and cell transformation. Point mutations at several other carboxyl-terminal positions generated transformation-defective E5 proteins that formed a complex with the PDGF receptor and induced receptor tyrosine phosphorylation but did not induce PDGF receptor down-regulation. Either PDGF receptor activation is not sufficient for transformation of C127 cells or the receptors that are tyrosine phosphorylated in response to these mutant E5 proteins are not fully activated and therefore are not able to deliver a mitogenic signal.

Amino Acid Sequence↗
PubMed1995

Ligand-independent activation of the platelet-derived growth factor beta receptor: requirements for bovine papillomavirus E5-induced mitogenic signaling.

The E5 protein of bovine papillomavirus type 1 binds to and activates the endogenous platelet-derived growth factor (PDGF) beta receptor in fibroblasts, resulting in cell transformation. We have developed a functional assay to test the ability of PDGF beta receptor mutants to mediate a mitogenic signal initiated by the E5 protein. Lymphoid Ba/F3 cells are strictly dependent on interleukin-3 for growth, but coexpression of the wild-type PDGF beta receptor and the E5 or v-sis-encoded protein generated a mitogenic signal which allowed Ba/F3-derived cells to proliferate in the absence of interleukin-3. In these cells, the E5 protein bound to and caused increased tyrosine phosphorylation of both the mature and the precursor forms of the wild-type PDGF beta receptor. The tyrosine kinase activity of the receptor was necessary for E5-induced receptor tyrosine phosphorylation and mitogenic activity but not for complex formation with the E5 protein. In contrast, the PDGF-binding domain of the receptor was not required for complex formation with the E5 protein, E5-induced tyrosine phosphorylation or mitogenic activity, demonstrating that E5-mediated receptor activation is ligand independent. Analysis of receptor mutants lacking various combinations of tyrosine phosphorylation sites revealed that the E5 and v-sis-encoded proteins display similar requirements for signaling and suggested that the wild-type PDGF beta receptor can generate multiple independent mitogenic signals. Importantly, these mutants dissociated two activities of the PDGF beta receptor tyrosine kinase, both of which are required for sustained mitogenic signaling: (i) receptor autophosphorylation and creation of binding sites for SH2 domain-containing proteins and (ii) phosphorylation of substrates other than the receptor itself.

Animals↗
PubMed1994

Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes.

We describe codon cassette mutagenesis, a simple method of mutagenesis that uses universal mutagenic cassettes to deposit single codons at specific sites in double-stranded DNA. A target molecule is first constructed that contains a blunt, double-strand break at the site targeted for mutagenesis. A double-stranded mutagenic codon cassette is then inserted at the target site. Each mutagenic codon cassette contains a three base pair direct terminal repeat and two head-to-head recognition sequences for the restriction endonuclease Sapl, an enzyme that cleaves outside of its recognition sequence. The intermediate molecule containing the mutagenic cassette is then digested with Sapl, thereby removing most of the mutagenic cassette, leaving only a three base cohesive overhang that is ligated to generate the final insertion or substitution mutation. A general method for constructing blunt-end target molecules suitable for this approach is also described. Because the mutagenic cassette is excised during this procedure and alters the target only by introducing the desired mutation, the same cassette can be used to introduce a particular codon at all target sites. Each cassette can deposit two different codons, depending on the orientation in which it is inserted into the target molecule. Therefore, a series of eleven cassettes is sufficient to insert all possible amino acids at any constructed target site. Thus codon cassettes are 'off-the-shelf' reagents, and this methodology should be a particularly useful and inexpensive approach for subjecting multiple different positions in a protein sequence to saturation mutagenesis.

Base Sequence↗
PubMed1994

Absence of a reservoir of Epstein-Barr virus (EBV) in normal tongue epithelium.

We examined human tongue epithelium and serum samples at autopsy for evidence of latent Epstein-Barr virus (EBV) infection. Although clinical serology revealed anti-EBV antibodies in most sera indicating past EBV infection, we found no Epstein-Barr nuclear antigen (EBNA)-coding sequences in tongue tissue by polymerase chain reaction (PCR), or Epstein-Barr-encoded RNA (EBER1) by in situ hybridization. Tongue epithelium does not appear to be a natural reservoir for latent EBV in immunocompetent hosts.

Adolescent↗
PubMed1994

Specific interaction between the bovine papillomavirus E5 transforming protein and the beta receptor for platelet-derived growth factor in stably transformed and acutely transfected cells.

The E5 protein of bovine papillomavirus is a 44-amino-acid membrane protein which induces morphologic and tumorigenic transformation of fibroblasts. We previously showed that the E5 protein activates and forms a complex with the endogenous beta receptor for platelet-derived growth factor (PDGF) in transformed rodent fibroblasts and that the PDGF beta receptor can mediate tumorigenic transformation by the E5 protein in a heterologous cell system. Other workers have identified the receptor for epidermal growth factor (EGF) as a potential target of the E5 protein in NIH 3T3 cells. Here, we investigate the specificity of the interaction of the E5 protein with various growth factor receptors, with particular emphasis on the PDGF beta receptor and the EGF receptor. Under conditions where both the PDGF beta receptor and the EGF receptor are stably expressed in E5-transformed mouse and bovine fibroblasts and in E5-transformed epithelial cells, the E5 protein specifically forms a complex with and activates the PDGF receptor and not the EGF receptor. Under conditions of transient overexpression in COS cells, the E5 protein has the potential to associate with several growth factor receptors, including the EGF receptor. However, upon coexpression of PDGF beta receptors and EGF receptors in COS cells, the E5 protein preferentially forms a complex with the PDGF receptor. Therefore, we conclude that the PDGF beta receptor is the primary target for the E5 protein in a variety of cell types, including bovine fibroblasts.

Animals↗
PubMed1993

Hierarchy of polyadenylation site usage by bovine papillomavirus in transformed mouse cells.

The great majority of viral mRNAs in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV) have a common 3' end at the early polyadenylation site which is 23 nucleotides (nt) downstream of a canonical poly(A) consensus signal. Twenty percent of BPV mRNA from productively infected cells bypasses the early polyadenylation site and uses the late polyadenylation site approximately 3,000 nt downstream. To inactivate the BPV early polyadenylation site, the early poly(A) consensus signal was mutated from AAUAAA to UGUAAA. Surprisingly, this mutation did not result in significant read-through expression of downstream RNA. Rather, RNA mapping and cDNA cloning experiments demonstrate that virtually all of the mutant RNA is cleaved and polyadenylated at heterogeneous sites approximately 100 nt upstream of the wild-type early polyadenylation site. In addition, cells transformed by wild-type BPV harbor a small population of mRNAs with 3' ends located in this upstream region. These experiments demonstrate that inactivation of the major poly(A) signal induces preferential use of otherwise very minor upstream poly(A) sites. Mutational analysis suggests that polyadenylation at the minor sites is controlled, at least in part, by UAUAUA, an unusual variant of the poly(A) consensus signal approximately 25 nt upstream of the minor polyadenylation sites. These experiments indicate that inactivation of the major early polyadenylation signal is not sufficient to induce expression of the BPV late genes in transformed mouse cells.

Animals↗
PubMed1993

Inhibition of cervical carcinoma cell line proliferation by the introduction of a bovine papillomavirus regulatory gene.

Human papillomavirus (HPV) E6 and E7 oncogenes are expressed in the great majority of human cervical carcinomas, whereas the viral E2 regulatory gene is usually disrupted in these cancers. To investigate the roles of the papillomavirus E2 genes in the development and maintenance of cervical carcinoma, the bovine papillomavirus (BPV) E2 gene was acutely introduced into cervical carcinoma cell lines by infection with high-titer stocks of simian virus 40-based recombinant viruses. Expression of the BPV E2 protein in HeLa, C-4I, and MS751 cells results in specific inhibition of the expression of the resident HPV type 18 (HPV18) E6 and E7 genes and in inhibition of cell growth. HeLa cells, in which HPV gene expression is nearly completely abolished, undergo a dramatic and rapid inhibition of proliferation, which appears to be largely a consequence of a block in progression from the G1 to the S phase of the cell cycle. Loss of HPV18 gene expression in HeLa cells is also accompanied by a marked increase in the level of the cellular p53 tumor suppressor protein, apparently as a consequence of abrogation of HPV18 E6-mediated destabilization of p53. The proliferation of HT-3 cells, a human cervical carcinoma cell line devoid of detectable HPV DNA, is also inhibited by E2 expression, whereas two other epithelial cell lines that do not contain HPV DNA are not inhibited. Thus, a number of cervical carcinoma cell lines are remarkably sensitive to growth inhibition by the E2 protein. Although BPV E2-mediated inhibition of HPV18 E6 and E7 expression may contribute to growth inhibition in some of the cervical carcinoma cell lines, the BPV E2 protein also appears to exert a growth-inhibitory effect that is independent of its effects on HPV gene expression.

Bovine papillomavirus 1↗
PubMed1993

Platelet-derived growth factor receptor can mediate tumorigenic transformation by the bovine papillomavirus E5 protein.

We showed previously that the beta receptor for platelet-derived growth factor (PDGF) is constitutively activated in fibroblasts transformed by the 44-amino-acid bovine papillomavirus type 1 (BPV) E5 protein and that the E5 protein and the PDGF receptor exist in a stable complex in E5-transformed fibroblasts. On the basis of these results, we proposed that activation of the PDGF receptor by the BPV E5 protein generates a sustained proliferative signal, resulting in fibroblast transformation. In this study, we used a gene transfer approach to provide functional evidence that the PDGF receptor can mediate transformation by the E5 protein. We show that normal mouse mammary gland (NMuMG) cells, a murine mammary epithelial cell line that does not express PDGF receptors, are not susceptible to transformation by the E5 protein. Coexpression of the PDGF beta receptor and E5 genes in these cells results in markedly increased tyrosine phosphorylation of an immature PDGF receptor species and the formation of a stable complex between the E5 protein and this immature PDGF receptor form. Importantly, introduction of the PDGF receptor gene into NMuMG cells renders them highly susceptible to E5-mediated tumorigenic transformation. In contrast, the E5 protein does not induce transformation via the endogenous epidermal growth factor receptor pathway in these cells. These results demonstrate that the PDGF receptor, a cellular protein with a well-characterized role in the positive control of cell proliferation, can mediate transformation by a DNA virus transforming protein.

Animals↗
PubMed1992

Stable association between the bovine papillomavirus E5 transforming protein and activated platelet-derived growth factor receptor in transformed mouse cells.

The 44-amino acid E5 transforming protein of bovine papillomavirus is the shortest protein known to induce tumorigenic transformation of fibroblasts. We showed previously that expression of the E5 protein activates the cellular beta receptor for platelet-derived growth factor (PDGF) and proposed that the activated receptor transmits the transforming signal to the cell. Here we use coimmunoprecipitation analysis to show that the E5 protein and the activated PDGF receptor exist in a stable complex in transformed mouse C127 cells. These results suggest a distinct mechanism of growth factor receptor activation and provide further evidence that the PDGF receptor is an important target of the E5 protein.

Animals↗
PubMed1992

Localization of bovine papillomavirus type 1 E5 protein to transformed basal keratinocytes and permissive differentiated cells in fibropapilloma tissue.

We examined expression of the E5 transforming protein of bovine papillomavirus type 1 (BPV-1) in naturally and experimentally infected bovine cells. Bovine conjunctival fibroblasts transformed in vitro by experimental infection with purified BPV-1 virions expressed significantly higher amounts of the 7-kDa E5 protein than BPV-1-transformed murine C127 cells. Indirect immunofluourescence analysis revealed a cytoplasmic, predominantly juxtanuclear, localization of E5 protein in the in vitro virus-transformed bovine cells. In naturally infected bovine skin fibropapilloma tissue, two widely separated sites of E5 protein synthesis were identified within the epithelial layers. Transformed basal layer keratinocytes throughout the tumor tissue expressed cytoplasmic E5 protein at a low uniform level. In addition, abundant amounts of cytoplasmic E5 protein with a granular staining pattern were detected in highly differentiated keratinocytes in close association with sites of viral capsid protein synthesis. These observations imply roles for the viral E5 oncogene in the growth transformation of basal epidermal keratinocytes as well as in the differentiation-linked process of viral maturation. Detection of a papillomavirus protein in the basal cell population of warts lends support to the hypothesis that these cells are maintained in a transformed state by continuous expression of a viral transforming gene.

Amino Acid Sequence↗
PubMed1992

The central hydrophobic domain of the bovine papillomavirus E5 transforming protein can be functionally replaced by many hydrophobic amino acid sequences containing a glutamine.

The 44-amino-acid E5 transforming protein of bovine papillomavirus can induce growth transformation of cultured rodent fibroblast cell lines. Previous studies revealed that efficient transformation of mouse C127 cells by the E5 protein required a central core of hydrophobic amino acids and several specific carboxyl-terminal amino acids. Although a randomly derived sequence of hydrophobic amino acids could functionally replace the wild-type hydrophobic core, most such sequences could not. We show here that the conserved glutamine at position 17 in the hydrophobic domain is also important for transformation and that insertion of the glutamine can rescue the transforming activity of many but not all otherwise defective mutants containing random hydrophobic sequences. However, a class of mutants was identified that transform efficiently even in the absence of glutamine, demonstrating that the presence of this amino acid is not absolutely required for efficient transformation. E5 proteins containing the glutamine appear to display increased homodimer formation compared with mutant proteins lacking the glutamine, but this amino acid has no apparent effect on protein stability.

Amino Acid Sequence↗
PubMed1992

The putative E5 open reading frame of cottontail rabbit papillomavirus is dispensable for papilloma formation in domestic rabbits.

In the cottontail rabbit papillomavirus (CRPV)-rabbit system, recombinant CRPV DNA can induce papillomas. This investigation was undertaken to evaluate whether the E5 open reading frame (ORF) of CRPV is required for papilloma formation. The CRPV genome we utilized, CRPV-WA, was sequenced in the E5 region and was found to contain one deletion, two insertions, and one transition mutation compared with CRPV-KS, the CRPV genome that has been fully sequenced. Despite these differences, an intact E5 ORF is preserved, supporting the notion that this gene may serve a biological function. One frameshift and two in-frame mutations were constructed in the small region of the 5' end of the E5 ORF that follows the E2 stop codon and precedes the L2 ORF. Several hundred rabbit skin sites were inoculated with each DNA preparation with a jet injector to test the ability of three CRPV E5 mutant DNAs to induce papillomas. In vivo results showed that each of the mutants induced papillomas, and biochemical analysis demonstrated that the E5 mutations present in DNA inocula were retained in the papillomas. The frequency of papilloma formation, however, was generally lower with each of the CRPV E5 mutants than with wild-type CRPV DNA, particularly so for the E5 frameshift mutant, suggesting that although the recognized E5 ORF is not required in domestic rabbits for the induction of papillomas by CRPV DNA, it may facilitate their formation.

Amino Acid Sequence↗
PubMed1991

Activation of the platelet-derived growth factor receptor by the bovine papillomavirus E5 transforming protein.

The bovine papillomavirus E5 gene encodes a 44 amino acid membrane-associated protein that can induce tumorigenic transformation of rodent fibroblast cell lines. Genetic studies suggest that the E5 protein may transform cells by influencing the activity of cellular proteins involved in growth regulation. We report here that the endogenous cellular beta type receptor for the platelet-derived growth factor (PDGF) is constitutively activated in C127 and FR3T3 cells stably transformed by the E5 protein, but not in these cell types transformed by a variety of other oncogenes. In C127 cells, a metabolic precursor as well as the mature form of the receptor is activated by E5 transformation. Activation of the receptor also occurs upon acute E5-mediated transformation of these cells and precedes mitogenic stimulation in this system. Moreover, activation of the receptor by addition of PDGF or the v-sis gene to untransformed cells is sufficient to induce DNA synthesis and stable growth transformation. We propose that the PDGF receptor is an important cellular intermediate in the transforming activity of the bovine papillomavirus E5 protein. There is a short region of sequence similarity between the fibropapillomavirus E5 proteins and PDGF, suggesting that the E5 proteins may activate the PDGF receptor by binding directly to it.

Animals↗