To date, all naturally occurring retinoblastoma susceptibility gene (RB) mutations known to be compatible with stable protein expression map to the T/E1A and cellular protein-binding region (the "pocket" domain). This domain extends from residue 379 to 792. When full-length RB and certain truncated forms were synthesized in human RB -/-cells, we found that the minimal region necessary for overt growth suppression extended from residue 379 to 928. A functional pocket domain and sequences extending from the carboxy-terminal boundary of the pocket to the carboxyl terminus of the protein were both necessary for growth suppression. Both sets of sequences were also required for E2F binding; hence, the two functions may be linked.

) cells causes a G 1 arrest and characteristic cellular swelling. Coexpression of the cellular transcription factor E2F-1 could overcome these effects. The ability of E2F-1 to bind to RB was neither necessary nor sufficient for this effect, and S-phase entry was not accompanied by RB hyperphosphorylation under these conditions. Furthermore, E2F-1 could overcome the actions of a nonphosphorylatable but otherwise intact RB mutant. These data, together with the fact that RB binds to E2F-1 in vivo, suggest that E2F-1 is a downstream target of RB action. Mutational analysis showed that the ability of E2F-1 to bind to DNA was necessary and sufficient to block the formation of large cells by RB, whereas the ability to induce S-phase entry required a functional transactivation domain as well. Thus, the induction of a G 1 arrest and the formation of large cells by RB in these cells can be genetically dissociated. Furthermore, the ability of the E2F-1 DNA-binding domain alone to block one manifestation of RB action is consistent with the notion that RB-E2F complexes actively repress transcription upon binding to certain E2F-responsive promoters. In keeping with this view, we show here that coproduction of an E2F1 mutant capable of binding to DNA, yet unable to transactivate, is sufficient to block RB-mediated transcriptional repression.

We show here that mammalian site-specific recombination and DNA-repair pathways share a common factor. The effects of DNA-damaging agents on cell lines derived from mice homozygous for the scid (severe combined immune deficiency) mutation were studied. Surprisingly, all scid cell lines exhibited a profound hypersensitivity to DNAdamaging agents that caused double-strand breaks (xirradiation and bleomycin) but not to other chemicals that caused single-strand breaks or cross-links. Neutral filter elution assays demonstrated that the x-irradiation hypersensitivity could be correlated with a deficiency in repairing double-strand breaks. These data suggest that the scid gene product is involved in two pathways: DNA repair of random doublestrand breaks and the site-specific and lymphoid-restricted variable-(diversity)-joining [V(D)J] DNA rearrangement process. We propose that the scid gene product performs a similar function in both pathways and may be a ubiquitous protein.Mice homozygous for the scid (severe combined immune deficiency) mutation lack a functional immune system but otherwise appear normal (1). The absence of B and T lymphocytes in scid mice is due to a defect in the site-specific V(D)J recombination pathway that is responsible for the somatic assembly of immunoglobulin and T-cell receptor genes. Analysis of scid variable (diversity) joining [V(D)J] recombination events has shown that large deletions, which remove all or most of the coding sequences of immunoglobulin or T-cell receptor genes, accompany the rearrangements and result in nonfunctional lymphoid cells (2-4). Furthermore, examination of model rearrangement templates (recombinant retroviruses and plasmids) introduced into scid lymphoid cells has recapitulated the aberrant deletional rearrangements (4-6). Thus, we and others have proposed that the scid gene product is an integral component of the V(D)J recombinase complex.Mutations that affect site-specific and general recombination frequently also affect the pathways responsible for repairing DNA double-strand breaks (DSBs) due to chromosome damage (7). This overlap of recombination and DSBrepair pathways is presumed to result from the postulated role of double-stranded ends as structural intermediates in many types of recombination and repair (for review, see ref.8). DSBs can also be generated by a number of DNAdamaging agents, the most common of which is ionizing radiation. X-ray-induced DSBs can stimulate chromosomal deletions and aberrant rearrangements and are lethal if not repaired (9). We show here that the similarity between recombination and DSB-repair pathways extends to mammalian cells affected by the scid mutation. scid cells were found to be hypersensitive specifically to agents that make DSBs. In addition, a dynamic assay for DNA repair demonstrated that the scid mutation severely diminished DSB repair. We propose that the scid gene product performs a similar function in both the V(D)J recombination and DSBrepair pathways. MATERIALS AND METHODSFibroblastic Cell Lines. Fib...

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-␤ has potent antiproliferative activity against most human tumor cells in vitro in addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-␤ concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here that ex vivo IFN-␤ gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Direct in vivo IFN-␤ gene delivery into established tumors generated high local concentrations of IFN-␤, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-␤ gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.