The p53 tumor suppressor gene and members of the transforming growth factor-␤ (TGF-␤) superfamily play central roles in signaling cell cycle arrest and apoptosis (programmed cell death) in normal development and differentiation, as well as in carcinogenesis. Here we describe a distantly related member of the TGF-␤ superfamily, designated placental TGF-␤ (PTGF-␤), that is upregulated in response to both p53-dependent and -independent apoptotic signaling events arising from DNA damage in human breast cancer cells. PTGF-␤ is normally expressed in placenta and at lower levels in kidney, lung, pancreas, and muscle but could not be detected in any tumor cell line studied. The PTGF-␤ promoter is activated by p53 and contains two p53 binding site motifs. Functional studies demonstrated that one of these p53 binding sites is essential for p53-mediated PTGF-␤ promoter induction and specifically binds recombinant p53 in gel mobility shift assays. PTGF-␤ overexpression from a recombinant adenoviral vector (AdPTGF-␤) led to an 80% reduction in MDA-MB-468 breast cancer cell viability and a 50 -60% reduction in other human breast cancer cell lines studied, including MCF-7 cells, which are resistant to growth inhibition by recombinant wild-type p53. Like p53, PTGF-␤ overexpression was seen to induce both G 1 cell cycle arrest and apoptosis in breast tumor cells. These results provide the first evidence for a direct functional link between p53 and the TGF-␤ superfamily and implicate PTGF-␤ as an important intercellular mediator of p53 function and the cytostatic effects of radiation and chemotherapeutic cancer agents.

We have mapped conserved regions of enhanced DNase I accessibility within the endogenous chromosomal locus of vascular endothelial growth factor A (VEGF-A). Synthetic zinc finger protein (ZFP) transcription factors were designed to target DNA sequences contained within the DNase I-hypersensitive regions. These ZFPs, when fused to either VP16 or p65 transcriptional activation domains, were able to activate expression of the VEGF-A gene as assayed by mRNA accumulation and VEGF-A protein secretion through a range exceeding that induced by hypoxic stress. Importantly, multiple splice variants of VEGF-A mRNA with defined physiological functions were induced by a single engineered ZFP transcription factor. We present evidence for an enhanced activation of VEGF-A gene transcription by ZFP transcription factors fused to VP16 and p65 targeted to two distinct chromosomal sites >500 base pairs upstream or downstream of the transcription start site. Our strategy provides a novel approach for dissecting the requirements for gene regulation at a distance without altering the DNA sequence of the endogenous target locus.

Targeting therapeutic gene expression to tumor cells represents a major challenge for cancer gene therapy. The strong transcriptional response exhibited by heat shock genes, along with the beneficial therapeutic effects of hyperthermia have led us to develop a heatdirected gene -targeting strategy for cancer treatment. Heat shock gene expression is mediated in large part by the interaction of heat shock factor 1 with specific binding sites ( heat shock elements; HSE ) found in the promoters of heat -inducible genes. Here we present a quantitative analysis of heat -inducible gene expression mediated by the wild -type hsp70b gene promoter, as well as a modified hsp70b promoter containing additional HSE sequences. -Galactosidase ( -gal ) expression was induced between 50 -and 800 -fold in a panel of human breast cancer cell lines infected with an adenoviral vector containing the wild -type hsp70b promoter ( Ad.70b. g ) following treatment at 438C for 30 minutes. Infection with an adenoviral vector containing the modified hsp70b promoter ( Ad.HSE.70b. g ) resulted in a 200 -to 950 -fold increase in -gal expression under the same conditions, and also provided a 1 ± 28C decrease in the threshold of activation. Significant increases in the heat responsiveness of the Ad.HSE.70b. g construct were observed in five of six tumor cell lines tested, as well as under thermotolerant conditions. Finally, we demonstrate that localized heating of a HeLa cell xenograft can effectively target -gal gene expression following intratumoral injection of Ad.70b. g. Adenoviral vectors incorporating heat -inducible therapeutic genes may provide useful adjuncts for clinical hyperthermia. Cancer Gene Therapy ( 2000 ) 7, 1566 ± 1574

Previous studies in our laboratory and others identified placental transforming growth factor-␤ (PTGF-␤) as an important downstream mediator of DNA damage signaling and a transcriptional target of p53. Here we show that accumulation of PTGF-␤ mRNA in response to p53 overexpression is delayed relative to p21 WAF1 , whereas the promoters of these genes respond to p53 with similar kinetics. Mutational analyses of two p53 binding sites within the PTGF-␤ promoter revealed that site p53-1 (؉29 bp) is responsible for as much as 80% of the transcriptional response to p53. This is consistent with electrophoretic mobility shift assays showing that site p53-1 binds p53 with a much higher affinity than site p53-2 (؊850 bp). We also describe for the first time a novel 21-bp element (؊222 to ؊242 bp) that acts to down-regulate the PTGF-␤ promoter response to p53. Termed the p53 transcriptional repressor element (p53TRE), this sequence was shown to suppress p53 transactivation in a position-and promoter-independent fashion and to associate with a 28-kDa protein expressed in several tumor cell lines. A p53 suppressor element and asymmetric p53 binding sites may participate determining the activation thresholds of p53-responsive promoters in a cell-and context-specific manner.