Specific N-methylations of HPV-16 E7 peptides alter binding to the retinoblastoma suppressor protein.

Complex formation between the human papilloma virus type 16 E7 protein (HPV-16 E7) and the retinoblastoma growth suppressor protein (RB) is believed to contribute to the process of cellular transformation that leads to cervical carcinoma. Genetic analysis of the HPV-16 E7 protein has shown that the segment of E7 homologous to the conserved region 2 of adenovirus 5 E1A protein is involved in both RB binding and E7-mediated cell transformation. We have previously shown that a peptide colinear with HPV-16 E7 residues 21-29 was able to block immobilized species of E7 from binding to RB protein. The current study reports the effects of different chemical modifications of this peptide. One type of modification, methylation of the alpha-amino nitrogens contributed by Leu22, Tyr25, and Leu28, resulted in a 45-fold increase in E7/RB binding antagonist activity. This increased antagonist activity is sequence-specific since methylation of the amino groups contributed by Tyr23, Cys24, or Glu26 resulted in a profound loss of binding antagonist activity. Using a newly developed binding assay we determined that the apparent dissociation constant for recombinant HPV-16 E7 protein binding to recombinant human RB protein is 1.3 nM. The peptide Ac[N-MeLeu22,N-Me-Tyr25,N-MeLeu28]-(21-29)-E7 amide was determined to be a competitive inhibitor of HPV-16 E7 binding to RB with a Ki value of 32 nM.

Mammalian cell lines engineered to identify inhibitors of specific signal transduction pathways.

A variety of signal transduction pathways contribute to the regulation of transcription in mammalian cells. Several of these pathways ultimately rely upon the interaction of transcription factors with genetic sequences termed response elements in the promoter regions of some genes. The biochemical mechanisms that control the levels and state of activation of transcription factors are poorly understood. However, specific phosphorylation events mediated by protein kinase C, growth factor receptor-linked tyrosine kinases, and protein kinase A clearly participate in the regulation of these signal transduction pathways. To understand the relationship between activation and/or inhibition of these pathways and regulation of gene expression controlled by specific response elements, cell lines were prepared containing the TPA response element (TRE), serum response element (SRE), or cyclic AMP response element (CRE) fused to a gene encoding a secretable form of alkaline phosphatase (SEAP). These TRE-SEAP, SRE-SEAP, and CRE-SEAP cells exhibit dramatic increases in alkaline phosphatase (AP) activity following exposure to TPA, PDGF, or forskolin. Down regulation of protein kinase C or inhibition of tyrosine kinase activity blocked the stimulation of AP activity caused by TPA or PDGF. These cell lines can be used to characterize existing inhibitors, and to identify new agents that affect specific signal transduction pathways in mammalian cells.

Lovastatin selectively inhibits ras activation of the 12-O-tetradecanoylphorbol-13-acetate response element in mammalian cells.

To evaluate ras-mediated signal transduction, an alkaline phosphatase gene (SEAP) was placed under the control of the ras-inducible phorbol ester response element (TRE) in murine fibroblasts (TRE-SEAP cells). The Kirsten ras gene was placed under the control of the glucocorticoid-inducible mouse mammary tumor virus promoter and introduced into the TRE-SEAP cells. Dexamethasone increased ras expression in the TRE-SEAP cells carrying the Kirsten ras gene and stimulated SEAP activity 25-fold. Lavostatin blocked dexamethasone induction of SEAP activity (50% inhibitory concentration, 0.5 microM) but did not affect phorbol ester-induced SEAP activity in the same cells. Lovastatin also did not block forskolin induction of SEAP activity in cells expressing SEAP under the control of the cyclic AMP response element.

Identification of HPV-16 E7 peptides that are potent antagonists of E7 binding to the retinoblastoma suppressor protein.

Complex formation between the human papilloma virus type-16 E7 protein (HPV-16 E7) and the retinoblastoma suppressor protein (pRB) is believed to be important in the process of cellular transformation that leads to cervical carcinoma. Utilizing an in vitro solution assay as well as a plate binding assay that measures the association between HPV-16 E7 and pRB proteins, we have examined a series of synthetic HPV-16 E7 peptides. HPV-16 E7 peptides which lie between amino acid residues 14 and 32 were found to be potent inhibitors of E7/pRB binding. The minimal peptide structure that possessed full antagonist activity was N-acetyl-E7-(21-29)-peptide amide. This peptide inhibited 100% of E7/pRB binding and exhibited an IC50 of 40 nM in the plate binding assay. A purified beta-galactosidase-E7 fusion protein exhibited an IC50 of 2 nM in the same assay. These results suggest that other regions of the E7 molecule in addition to amino acids 21-29 may contributed to E7/pRB interaction. Analysis of E7-(20-29)-peptides containing single amino acid substitutions suggests that Cys24, Tyr23, Tyr25, Asp21, and Glu26 are important residues for maintaining maximal antagonist activity. This series of peptides should prove useful in analyzing the biological consequences of E7/pRB binding in HPV-infected cells.

Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.

Transforming growth factor-alpha (TGF-alpha) is a growth-promoting protein that binds to the epidermal growth factor (EGF) receptor. To identify critical residues that govern TGF-alpha-EGF receptor binding, we prepared site-specific substitution mutants of TGF-alpha. Mutant proteins were tested in receptor-binding and mitogenesis assays. Semiconservative substitutions at positions 4, 12, 18, and 45 decreased biological activity 2.1- to 14-fold. The conservative substitution of lysine for arginine at position 42 completely eliminated biological activity. Amino acid composition analysis of proteolytic fragments from TGF-alpha and the Lys-42 mutant indicated that these proteins contained the same disulfide bonds. These studies suggest that arginine 42 may be a contact point for TGF-alpha-EGF receptor interaction.

Spontaneously transformed NRK cells lose their mitogenic response to epidermal growth factor.

To understand the relationship between growth factor-induced mitogenesis and spontaneous cell transformation, a clonal isolate of epidermal growth factor (EGF)-responsive NRK cells was passed in vitro until morphologically transformed variants arose. Subclones of EGF responsive (Cl-3) and EGF nonresponsive (Cl-10) NRK cells were isolated. Cl-3 cells grew as flat, contact-inhibited monolayers, while Cl-10 cells grew as rounded or spindle-shaped cells that formed dense foci. Cl-10 cells formed colonies in soft agar more efficiently (p less than 0.01) and formed larger tumors in nude mice (p less than 0.05) than Cl-3 cells. Cl-3 cells exhibited a sixfold increase in DNA synthesis in response to 1.0 nM EGF. Cl-10 cells did not increase DNA synthesis on exposure to 100 nM EGF. These different responses to EGF occurred despite similar numbers of receptors and similar receptor.binding affinities for EGF (Cl-3: 7000 receptors, Kd = 0.67 nM; Cl-10: 8000 receptors, Kd = 0.72 nM). No evidence of transforming growth factor-alpha was detected in either of these cell lines using Northern blots, Western blots, or biologic assays. We conclude that NRK cells which undergo spontaneous morphologic transformation and exhibit enhanced anchorage-independent growth lose their mitogenic response to EGF.

Structure-function analysis of synthetic and recombinant derivatives of transforming growth factor alpha.

Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors. To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha. These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays. Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity. Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins. However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity. Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis. However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold). These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity.

Isolation of multiple biologically and chemically diverse species of epidermal growth factor.

We have analyzed several lots of epidermal growth factor (EGF) purified from murine submaxillary glands including "receptor grade" EGF from Collaborative Research and EGF from Boehringer Mannheim Biochemicals. New England Nuclear uses "receptor grade" EGF to produce 125I-labeled EGF. Though these reagents are reported to be homogeneous, we found them to be a mixture of six species. A method was developed to separate this mixture into its component parts. The individual components were chemically characterized and tested for biological potency. N-terminal sequence analysis of the unfractionated EGF-mixture reveals three different sequences starting with residues 1, 2, or 3 of the mature peptide. Each component exhibited different degrees of mitogenic and EGF receptor binding activity indicating that the N-terminal region contributes to the biological response. The species representing the complete EGF peptide is the most active species in all biological assays. A rapid method for purification of homogeneous complete EGF from commercial EGF preparations is described.

Canine infectious tracheobronchitis: effects of an intranasal live canine parainfluenza-Bordetella bronchiseptica vaccine on viral shedding and clinical tracheobronchitis (kennel cough).

A modified-live intranasal (IN) canine parainfluenza (CPI)-virus Bordetella bronchiseptica vaccine was evaluated in dogs for efficacy against laboratory-induced canine infectious tracheobronchitis. The comparative efficacies of IN and parenteral administrations of the CPI virus fraction were also evaluated. The frequency and duration of clinical tracheobronchitis, blood serum agglutination titer, humoral antibody response, and duration of CPI virus and B bronchiseptica shedding were measured. Group A dogs were vaccinated subcutaneously or IM with an experimental CPI vaccine and challenge exposed with CPI virus. Group B dogs were vaccinated IN with avirulent CPI virus-B bronchiseptica live antigens and challenge exposed with virulent CPI virus and virulent B bronchiseptica. The IN vaccination (group B) significantly reduced (P less than or equal to 0.001) the occurrence of clinical tracheobronchitis by 96%. The combined challenge exposure of virulent CPI and virulent B bronchiseptica produced a synergistic enhancement of the clinical signs of kennel cough. The percentage of days after challenge exposure that virus shedding was detected for controls equaled 70% as compared with 50% and only 1% for parenterally and IN vaccinated dogs, respectively. Isolation of virulent B bronchiseptica microorganisms was reduced 89% in dogs vaccinated IN compared to controls. The geometric mean humoral antibody titers to CPI virus after 2 parenteral vaccinations and 1 IN vaccination were 1:43 and 1:34, respectively.

Protections by non-copper metal ions against copper-mediated inactivation of poliovirion RNA occurring at extraction of virions with phenol.

Al3+, Ca2+, Co2+, Cu1+, Fe2+, Fe3+, Mg2+, Mn2+, Ni2+, Pb2+, Sn4+, and Zn2+ were incubated individually with redistilled reagent-grade phenol containing impurities known from previous work to interact with Cu2+ to produce a potent inactivator(s) of the transfectivity of naked poliovirion RNA. Only the mixture with Cu1+ inactivated the RNA. Tests of each of the 11 non-copper test metal ions mixed with Cu2+ before adding the phenol showed that Ca2+ and Mg2+ do not protect, Co2+, Mn2+, Ni2+, Pb2+, and Zn2+ provide moderate protection, and Al3+, Fe2+, Fe3+, and Sn4+ give strong protection against the Cu2+-mediated inactivation. Other points of addition of protective metal ion were tested using Fe3+. Strong protection was afforded even when Fe3+ was added after synthesis of the inactivator(s) from Cu2+ and the active impurities. The relation between Cu2+ and the Fe3+ was shown to be competitive. The hypothesis that ions compete for semi-quinone anion is proposed.

Inactivation of transfective poliovirion RNA by a product or products of the interaction of trace copper with an impurity or impurities in reagent-grade phenol.

A search for the cause of the inactivation of the transfectivity of the RNA from poliovirions, in the absence of a protective agent such as L-histidine, revealed that the inactivation is associated with trace contamination with copper and with an impurity or impurities in the phenol used to release the RNA from the poliovirions. Cu2+ and the impurity(ies) interact in vitro to produce a proximate inactivator or inactivators of the RNA. Phenol free or nearly free of active impurity can be prepared by steam distillation. Light is not required for formation or for action of the proximate inactivator. Addition of L-histidine to RNA undergoing inactivation promptly stops the inactivation, probably by taking copper away from the proximate inactivator.

Influence of polypeptide molecular and side chain lengths and of side chain steric location on the kinetics of basic polypeptide-induced sensitization of primate cells to transfection.

Kinetics of sensitization of chimpanzee cell sheets to transfection by poliovirus RNA was determined for 5 basic polypeptides. With basic olypeptide hydrobromide at 100 microng/ml, initial sensitization rate was faster for poly-L-ornithine of average molecular weight (AMW) 15500 than of AMW 105000, and much faster for poly-L-lysine of AMW 1700 than of AMW 140000. Desensitization phases were observed with the 2 shorter polypeptides. Sensitization was much faster and sensitivity maxima were considerably higher for the polyornithines than for the polylysines. Poly-D-lysine of AMW 160000 sensitized cells slightly faster than poly-L-lysine of AMW 140000, but gave about the same sensitivity maximum. Analysis of the slow cell sensitization by poly-L-lysine of AMW 140000 revealed 2 steps: a fast step 1 (attachment) and a slow step 2 (processing).