Targeting DNA to cells with basic fibroblast growth factor (FGF2).

Ligand-mediated targeting of DNA was validated by condensing a plasmid DNA encoding the beta-galactosidase (beta-gal) gene with a basic fibroblast growth factor (FGF2) that was first chemically conjugated to polylysine (K). The conditions that gave optimal binding of this FGF2 to DNA also generated the highest level of beta-gal expression when added to FGF2 target cells like COS-1, 3T3, baby hamster kidney (BHK), or endothelial cells. This beta-gal activity increased in a time- and dose-dependent manner and was dependent on the inclusion of FGF2 in the complex. FGF receptor specificity was demonstrated by competition of the complex with FGF2 and heparin, and by the failure of cytochrome c or histone H1 to mimic the gene-targeting effects of FGF2. The expression of beta-gal was also endosome dependent because chloroquine increased beta-gal expression 8-fold and endosome disruptive peptides increased expression of beta-gal 26-fold. Taken together these findings establish that DNA can be introduced into cells through the high affinity FGF receptor complex, and while its efficiency will require significant enhancements to achieve sustained and elevated transgene expression, the possibility that the technique could be used to deliver DNAs encoding cytotoxic molecules is discussed.

Synthesis and characterization of a homogeneous chemical conjugate between basic fibroblast growth factor and saporin.

Basic fibroblast growth factor (FGF-2) and saporin were chemically conjugated using the crosslinker, N-succinimidyl-3(2-pyridyldithio)-propionate. When purified, the conjugate was found to be heterogeneous as analyzed by SDS/PAGE, size-exclusion HPLC and reverse-phase HPLC. Therefore, we sought to synthesize a molecule that would be homogeneous and thus easier to characterize and evaluate its efficacy and toxicity for pharmaceutical drug development. A homogeneous chemical conjugate was successfully synthesized by using a mutant FGF-2 with Cys96 replaced by Ser ([S96]FGF-2) and a recombinant saporin mutant containing a single Cys at the -1 position (C-SAP). The latter was expressed in Escherichia coli and isolated to 99% purity by expanded-bed adsorption chromatography followed by cation-exchange chromatography. The cysteine in C-SAP was activated by Ellman's reagent and then reacted with the only available cysteine (position 78) in [S96]FGF-2 to produce the homogeneous conjugate, designated as FGF2-C-SAP. The purified FGF2-C-SAP was more than 98% pure as judged by HPLC. In vitro biological assays indicated that FGF2-C-SAP was a potent inhibitor of protein synthesis in a cell-free system and was cytotoxic to FGF-receptor-bearing cells.

Regulation-defective mutants of type I cAMP-dependent protein kinase. Consequences of replacing arginine 94 and arginine 95.

The type I alpha regulatory subunits of cAMP-dependent protein kinase contain an autoinhibitor site, Arg94-Arg-Gly-Ala-Ile, which serves as a pseudosubstrate. To evaluate their contribution to subunit association, Arg94 and Arg95, key determinants for peptide recognition, were replaced singly and in tandem with Ala, Glu, and His. Unlike substrate peptides in which replacement of either arginine leads to an increase in Km of approximately 3 orders of magnitude, replacement of either arginine causes only a maximal 20-fold decrease in subunit association. Replacement of both arginine residues with alanine, however, generates a regulatory subunit that can no longer recombine with the catalytic subunit under physiological conditions when the regulatory subunit is saturated with cAMP. To evaluate more fully the specific consequences of replacing these 2 arginine residues, a rapid gel filtration chromatographic method was developed so that subunit affinity could be measured independently of assaying for catalytic activity. The R94,95A mutant shows a Kd(app) = 677 nM, representing an increase of greater than 3 orders of magnitude compared with the native subunits in the presence of MgATP. In the absence of MgATP, the Kd(app) for native regulatory subunit was 125 nM, whereas the Kd(app) for the R94,R95A mutant regulatory subunit was determined to 2.87 microM. When this mutant holoenzyme is assayed at microM concentrations, no activity is observed, whereas below microM, activity is observed because of cAMP-independent subunit dissociation.

Mutations in the autoinhibitor site of the regulatory subunit of cAMP-dependent protein kinase I. Replacement of Ala-97 and Ser-99 interferes with reassociation with the catalytic subunit.

Each regulatory (R) subunit of cAMP-dependent protein kinase contains an autoinhibitor site that lies approximately 90-100 residues from the amino terminus. In order to study the importance of this autoinhibitor site in the type I R-subunit for interacting with the catalytic (C) subunit, recombinant techniques were used to replace Ala-97 with Gln, His, Lys, and Arg and to replace Ser-99 with Gly and Lys. All of the mutant proteins having a replacement at Ala-97 showed reduced affinity for the C-subunit ranging from 14- to 55-fold. In general, the decrease in affinity of the Ala-97 mutants for the C-subunit correlated with the increase in size of the side chain. In contrast to wild type R-subunit, where MgATP facilitates holoenzyme formation, MgATP inhibits the reassociation in all of the Ala-97 mutants suggesting that the larger side chains sterically interfere with bound MgATP in the active site of the C-subunit. Whereas MgATP slowed holoenzyme formation, AMP actually accelerated the reassociation of the A97K, A97H (pH 6.0), and A97Q mutants with the C-subunit. Therefore, the side chains of Lys-97, His-97, and Gln-97 can interact either electrostatically or by hydrogen bonding with the phosphate of AMP. This interpretation is reinforced by the fact that the stimulatory effect of AMP on the A97H mutant was pH-dependent. The affinities of the S99G and S99K mutants for the C-subunit were reduced 7- and 24-fold, respectively, suggesting that Ser-99 also may contribute to interactions between the R- and C-subunits.

Fluorescence ratio imaging of cyclic AMP in single cells.

Fluorescence imaging is perhaps the most powerful technique currently available for continuously observing the dynamic intracellular biochemistry of single living cells. However, fluorescent indicator dyes have been available only for simple inorganic ions such as Ca2+, H+, Na+, K+, Mg2+ and Cl-. We now report a fluorescent indicator for the adenosine 3',5'-cyclic monophosphate (cAMP) signalling pathway. The sensor consists of cAMP-dependent protein kinase in which the catalytic (C) and regulatory (R) subunits are each labelled with a different fluorescent dye such as fluorescein or rhodamine capable of fluorescence resonance energy transfer in the holoenzyme complex R2C2. When cAMP molecules bind to the R subunits, the C subunits dissociate, thereby eliminating energy transfer. The change in shape of the fluorescence emission spectrum allows cAMP concentrations and the activation of the kinase to be nondestructively visualized in single living cells microinjected with the labelled holoenzyme.