Phosphorylation and active ATP hydrolysis are not required for SV40 T antigen hexamer formation.

ATP induces structural alterations in SV40 large T antigen and promotes changes in its interaction with the viral replication origin. We have analyzed nucleotide-induced changes in T antigen structure in the absence of origin DNA. Most preparations of immunopurified T antigen contain several discrete species ranging in size from monomers through oligomers larger than hexamers. The predominant species consist of monomers and dimers. Incubation of T antigen with ATP or dATP leads to a dramatic and rapid increase in the appearance of T antigen hexamers. Weakly and nonhydrolyzable analogs of ATP are effective as well, indicating that hexamer formation does not require active ATP hydrolysis. After incubation of T antigen with [gamma-35S]ATP, stable association of the labeled nucleotide with all detectable forms occurs. Removal of greater than 80% of the T antigen phosphate residues does not significantly affect the formation of T antigen hexamers, although changes in the distribution and mobility of the other species of T antigen are apparent. Furthermore, T antigen synthesized in and purified from Escherichia coli and, therefore, presumably un- or underphosphorylated, is capable of forming hexamers. Nucleotide-induced T antigen hexamer formation thus appears to require neither protein phosphorylation nor active ATP hydrolysis.

Antitumor activity of the single-chain immunotoxin BR96 sFv-PE40 against established breast and lung tumor xenografts.

We have constructed a single-chain immunotoxin consisting of the variable H and L chains of the carcinoma-reactive mAb BR96, fused to the binding defective protein toxin, PE40. This molecule, BR96 sFv-PE40, has been shown to be extremely cytotoxic toward a variety of BR96 Ag-expressing tumor cell lines. When administered i.v. into athymic mice carrying L2987 tumor xenografts, BR96 sFv-PE40 was cleared rapidly from the blood with a half-life of approximately 30 min. This is in comparison to a chemical conjugate, chiBR96-LysPE40, that remained in the blood for almost 2 h. In addition, the smaller single-chain immunotoxin (67 kDa) penetrates the tumor faster than the larger chemical conjugate (190 kDa). Using a variety of administration schedules and doses, we treated established human tumor xenografts in athymic mice with both the single-chain immunotoxin BR96 sFv-PE40 and the chemical conjugate chiBR96-LysPE40. In both L2987 lung carcinoma and MCF-7 breast carcinoma models, we found that BR96 sFv-PE40 completely regressed the tumor xenografts. With an administration schedule of q4dx5, the tumors were totally regressed and did not reappear. The chiBR96-LysPE40 conjugate produced partial tumor regressions, although at near maximum tolerated dose. These results show that the single-chain immunotoxin, BR96 sFv-PE40, is a potent antitumor agent.

The p53 protein is an unusually shaped tetramer that binds directly to DNA.

We have analyzed the size and structure of native immunopurified human p53 protein. By using a combination of chemical crosslinking, gel filtration chromatography, and zonal velocity gradient centrifugation, we have determined that the predominant form of p53 in such preparations is a tetramer. The behavior of purified p53 in gels and sucrose gradients implies that the protein has an extended shape. Wild-type p53 has been shown to bind specifically to sites in cellular and viral DNA. We show in this study by Southwestern ligand blotting and by analysis of DNA-bound crosslinked p53 that p53 monomers, dimers, and tetramers can bind directly to DNA.

BR96 sFv-PE40, a potent single-chain immunotoxin that selectively kills carcinoma cells.

We have constructed a single-chain immunotoxin composed of the carcinoma-reactive antibody BR96 and a truncated form of Pseudomonas exotoxin. The chimeric molecule, BR96 sFv-PE40, was expressed in Escherichia coli and localized to the inclusion bodies. We purified and identified two species of BR96 sFv-PE40, monomers and aggregates. The monomeric form was able to bind well to the BR96 antigen, a Lewisy-related antigen, while the aggregate was not. The binding affinity of the monomeric recombinant immunotoxin was 5-fold less than intact BR96 IgG, and its specificity for the BR96 antigen was confirmed by competition analysis. Monomeric BR96 sFv-PE40 was found to be extremely cytotoxic against cancer cells displaying the BR96 antigen. The cytotoxicity of the fusion protein correlates directly with antigen density on the tumor cell lines tested. The breast carcinoma cell line MCF-7, which has the highest density of BR96 antigen, was the most sensitive to BR96 sFv-PE40, with a concentration producing 50% protein synthesis inhibition of 5 pM. BR96 sFv-PE40 was found to have a t1/2 in serum of 28.5 min in athymic mice, compared to that of the chemical conjugate, chiBR96-LysPE40, which was 54 min. These data indicate that the single-chain immunotoxin BR96 sFv-PE40 is a potent inhibitor of protein synthesis in target cell lines and may be an effective agent for the treatment of cancer.

Site-specific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen and mutant p53.

Wild-type p53 protein was shown to bind specifically to DNA sequences within SV40 (Bargonetti et al. 1991), the human ribosomal gene cluster (RGC) (Kern et al. 1991a), and the murine muscle creatine kinase gene (MCK) (Zambetti et al. 1992). However, a direct comparison of these three sites was not performed. Here we demonstrate, by filter binding and gel mobility-shift assays, that wild-type p53 binds with similar affinities to MCK and RGC sites but less tightly to the SV40 site. We examined the effects of two candidate regulators of p53 function, SV40 large T antigen and oncogenic mutant p53, on the binding of wild-type p53 to RGC DNA. We show that wild-type T antigen prevents p53 from binding to the RGC site under all conditions tested. Moreover, two temperature-sensitive mutant SV40 T antigens, which fail to transform cells at the nonpermissive temperature, prevent p53 from binding to the RGC site at the permissive, but not at the restrictive, temperature. The ability of complexes containing wild-type p53 and tumor-derived mutant p53 proteins to bind to RGC DNA varies according to the position of the mutation. Complexes containing wild-type and either his175 or his273 mutant p53 proteins are completely unable to bind to the RGC DNA sequence. Interestingly, a complex containing wild-type p53 and the trp248 mutant p53 characteristic of Li-Fraumeni syndrome patients displays nearly wild-type levels of binding. Perhaps this mutant allele can be tolerated in these individuals because the wild-type mutant p53 complex maintains the ability to bind to DNA. Our data indicate that the oncogenic potential of both T antigen and some mutant p53 proteins is the result of their ability to block binding of wild-type p53 to DNA.

Murine p53 inhibits the function but not the formation of SV40 T antigen hexamers and stimulates T antigen RNA helicase activity.

We have characterized the effects of p53 on several biochemical activities of simian virus 40 (SV40) large tumor (T) antigen. While p53 induced a strong inhibition of the T antigen DNA helicase activity, surprisingly, its RNA helicase activity was stimulated. This supports the liklihood that the DNA and RNA helicase activities of T antigen reflect discrete functions. p53 did not significantly affect the ATP-dependent conversion of T antigen monomers to hexamers. However, the ability of these hexamers to assemble on a DNA fragment containing the viral origin was impaired by p53. Thus, these results suggest that p53 inhibits the function but not the formation of T antigen multimers. This conclusion was further supported by the observation that the addition of a purified p53:T antigen complex was as inhibitory as free p53 to the DNA helicase activity of free T antigen. Thus our data indicates that the targets of p53 inhibition are the functional units of T antigen, namely the hexamers.

Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication.

The DNA from a wide variety of human tumors has sustained mutations within the conserved p53 coding regions. We have purified wild-type and tumor-derived mutant p53 proteins expressed from baculovirus vectors and examined their interactions with SV40 DNA. Using DNAase I footprinting assays, we observed that both human and murine wild-type p53 proteins bind specifically to sequences adjacent to the late border of the viral replication origin. By contrast, mutant p53 proteins failed to bind specifically to these sequences. SV40 T antigen prevented wild-type p53 from interacting with this region. These data show that normal but not oncogenic forms of p53 are capable of sequence-specific interactions with viral DNA. Furthermore, they provide insights into the mechanisms by which viral proteins might regulate the control of viral growth and cell division.

Wild-type, but not mutant, human p53 proteins inhibit the replication activities of simian virus 40 large tumor antigen.

Murine p53 blocks many of the replication activities of simian virus 40 (SV40) large tumor antigen (T antigen) in vitro. As murine cells do not replicate SV40 DNA, it was of interest to determine how p53 from permissive human cells functions. Recombinant baculoviruses encoding either the wild-type form of human p53 or a mutant p53 cloned from a human tumor cell line were constructed, and p53 proteins were purified from infected insect cells. Surprisingly, we found that wild-type human p53 was as inhibitory to the ability of T antigen to mediate replication of an SV40 origin-containing (ori DNA) plasmid in vitro as was murine p53. Wild-type human p53 also blocked the DNA unwinding activity of T antigen, as did its murine counterpart. In contrast to murine and wild-type human p53, the mutant human p53 did not block ori DNA replication or DNA unwinding. Murine p53 formed a complex with mutant human p53 in vivo. Furthermore, mutant human p53 reduced the inhibition of SV40 ori DNA replication by murine p53 in vitro. These results provide a model for the way in which mutant p53 proteins can affect normal functions of p53.

Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2.

The human anti-oncoprotein p53 is shown to be a substrate of cdc2. The primary site of phosphorylation is serine-315. Serine-315 is phosphorylated by both p60-cdc2 and cyclin B-cdc2 enzymes. The phosphorylation of p53 is cell cycle-dependent. The abundance of p53 also oscillates during the cell cycle. The protein is largely absent from cells that have just completed division but accumulates in cells during G1 phase. Phosphorylation by cdc2 might regulate the antiproliferative activity of p53.

The murine p53 protein blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T antigen.

We have characterized the effect of murine p53 on SV40 DNA replication in vitro. Purified wild-type murine p53 dramatically inhibited the ability of SV40 T antigen to mediate the replication of a plasmid bearing the viral origin (ori-DNA) in vitro. In contrast, polyoma ori-DNA replication in vitro was unaffected by p53. Surprisingly, both unbound p53 and SV40 T antigen-bound p53 were equally detrimental to SV40 ori-DNA replication. Thus, p53 interferes with interactions between T antigen molecules that are required for DNA synthesis. p53 inhibited the binding to and subsequent unwinding of the SV40 origin by T antigen and thus selectively blocked the initial stages of ori-DNA replication. In contrast to the nononcogenic wild-type murine p53, high concentrations of a mutant transforming p53 failed to block SV40 ori-DNA replication in vitro. These observations may provide insight into a possible role for p53 in the cell.