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.

The largest known gene is the human dystrophin gene, which has 79 exons spanning at least 2,300 kilobases (kb). Transcript accumulation was monitored from four regions of the gene following induction of expression in muscle cell cultures. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) results indicate that approximately 12 h are required for transcription of 1,770 kb (at an average elongation rate of 2.4 kb min-1), extrapolating to a transcription time of 16 h for the complete gene. Accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5' end before transcription is complete providing strong evidence for cotranscriptional splicing. The rate of transcript accumulation was reduced at the 3' end of the gene relative to the 5' end, perhaps due to premature termination of transcription complexes.

Duchenne muscular dystrophy (DMD) and its milder form, Becker muscular dystrophy (BMD), are allelic X-linked muscle disorders in man. The gene responsible for the disease has been cloned from knowledge of its map location at band Xp21 on the short arm of the X chromosome. The product of the DMD gene, a protein of relative molecular mass 400,000 (Mr 400K) recently named dystrophin, has been reported to co-purify with triads of mouse and rabbit skeletal muscle when assayed using polyclonal antibodies raised against fusion proteins encoded by regions of mouse DMD complementary DNA. Here we show that antibodies directed against synthetic peptides and fusion proteins derived from the N-terminal region of human DMD cDNA strongly react with an antigen present in skeletal muscle sarcolemma on cryostat sections of normal human muscle biopsies. This immunoreactivity is reduced or absent in muscle fibres from DMD patients but appears normal in muscle fibres from patients with other myopathic diseases. The same antibodies specifically react with a 400K protein in sodium dodecyl sulphate (SDS) extracts of normal human muscle subjected to Western blot analysis. We conclude that the product of the DMD gene is associated with the sarcolemma rather than with the triads and speculate that it strengthens the sarcolemma by anchoring elements of the internal cytoskeleton to the surface membrane.

Duchenne muscular dystrophy (DMD) and its less severe form Becker muscular dystrophy (BMD) are allelic disorders. It has been suggested that in the mutations involving BMD, the translational reading frame of messenger RNA is maintained and a smaller, though partially functional, protein is produced. In order to test this, the exon-intron boundaries of the first ten exons of the DMD gene were determined, and 29 patients were analyzed. In a number of BMD patients (mild and severe BMD), the reading frame of messenger RNA was not maintained. On the basis of these findings, a model for reinitiation from an internal start codon is suggested.

No abstract

This study assessed the feasibility of using an ex vivo stem cell antigen-1-positive (Sca-1(+)) cell-based systemic fibroblast growth factor-2 (FGF-2) gene therapy to promote endosteal bone formation. Sca-1(+) cells were used because of their ability to home to, and engraft into, the bone marrow cavity. The human FGF-2 gene was modified to increase protein secretion and stability by adding the bone morphogenic protein (BMP)-2/4 hybrid signal sequence and by mutating two key cysteines. Retro-orbital injection of Sca-1(+) cells transduced with a Moloney leukemia virus (MLV)-based vector expressing the modified FGF-2 gene into sub-lethally irradiated W(41)/W(41) recipient mice resulted in long-term engraftment, more than 100-fold elevation in serum FGF-2 level, increased serum bone-formation markers, and massive endosteal bone formation. In recipient mice showing very high serum FGF-2 levels (>2,000 pg/ml), this enhanced endosteal bone formation was so robust that the marrow space was filled with bony tissues and insufficient calcium was available for the mineralization of all the newly formed bone, which led to secondary hyperparathyroidism and osteomalacia. These adverse effects appeared to be dose related. In conclusion, this study provided compelling test-of-principle evidence for the feasibility of using an Sca-1(+) cell-based ex vivo systemic FGF-2 gene therapy strategy to promote endosteal bone formation.

Previous studies have demonstrated that overexpression of GRP78/BiP, an endoplasmic reticulum (ER)-resident molecular chaperone, in mammalian cells inhibits the secretion of specific coagulation factors. However, the effects of GRP78/BiP on activation of the coagulation cascade leading to thrombin generation are not known. In this study, we examined whether GRP78/BiP overexpression mediates cell surface thrombin generation in a human bladder cancer cell line T24/83 having prothrombotic characteristics. We report here that cells overexpressing GRP78/BiP exhibited significant decreases in cell surface-mediated thrombin generation, prothrombin consumption and the formation of thrombin-inhibitor complexes, compared with wild-type or vector-transfected cells. This effect was attributed to the ability of GRP78/BiP to inhibit cell surface tissue factor (TF) procoagulant activity (PCA) because conversion of factor X to Xa and factor VII to VIIa were significantly lower on the surface of GRP78/BiP-overexpressing cells. The additional findings that (i) cell surface factor Xa generation was inhibited in the absence of factor VIIa and (ii) TF PCA was inhibited by a neutralizing antibody to human TF suggests that thrombin generation is mediated exclusively by TF. GRP78/BiP overexpression did not decrease cell surface levels of TF, suggesting that the inhibition in TF PCA does not result from retention of TF in the ER by GRP78/BiP. The additional observations that both adenovirus-mediated and stable GRP78/BiP overexpression attenuated TF PCA stimulated by ionomycin or hydrogen peroxide suggest that GRP78/BiP indirectly alters TF PCA through a mechanism involving cellular Ca 2؉ and/or oxidative stress. Similar results were also observed in human aortic smooth muscle cells transfected with the GRP78/BiP adenovirus. Taken together, these findings demonstrate that overexpression of GRP78/BiP decreases thrombin generation by inhibiting cell surface TF PCA, thereby suppressing the prothrombotic potential of cells.

No abstract