Loss of epithelial p53 and αv integrin cooperate through Akt to induce squamous cell carcinoma yet prevent remodeling of the tumor microenvironment.

Most of the squamous cell carcinomas (SCCs) of the skin and head and neck contain p53 mutations. The presence of p53 mutations in premalignant lesions suggests that they represent early events during tumor progression and additional alterations may be required for SCC development. Here we show that codeletion of the p53 and αv integrin genes in mouse stratified epithelia induced SCCs in 100% of the mice, more frequently and with much shorter latency than deletion of either gene alone. The SCCs that lacked p53 and αv in the epithelial tumor cells exhibited high Akt activity, lacked multiple types of infiltrating immune cells, contained a defective vasculature and grew slower than tumors that expressed p53 or αv. These results reveal that loss of αv in epithelial cells that lack p53 promotes SCC development, but also prevents remodeling of the tumor microenvironment and delays tumor growth. We observed that Akt inactivation in SCC cells that lack p53 and αv promoted anoikis. Thus, tumors may arise in these mice as a result of the increased cell survival induced by Akt activation triggered by loss of αv and p53, and by the defective recruitment of immune cells to these tumors, which may allow immune evasion. However, the defective vasculature and lack of a supportive stroma create a restrictive microenvironment in these SCCs that slows their growth. These mechanisms may underlie the rapid onset and slow growth of SCCs that lack p53 and αv.

Beta 3 integrins regulate lymphocyte migration and cytokine responses in heart transplant rejection.

Integrin alpha v beta 3 is important for cell survival, signaling and migration, particularly during angiogenesis and tumorigenesis, where it has been proposed as a therapeutic target. alpha v beta 3 is up-regulated following transplantation and beta 3 polymorphisms are associated with increased acute kidney rejection, suggesting that alpha v beta 3 may also play a role in transplant rejection. Here, using a model of allogeneic heart transplantation, we show that allograft survival is prolonged in beta 3 integrin-deficient (beta 3(-/-)) mice. This is associated with Th2-type immune responses and reduced T-cell infiltration into grafts and T cells from beta 3(-/-) mice show impaired adhesion and migration, consistent with a role for alpha v beta 3 in transmigration. These studies provide evidence that targeting beta 3 integrins impairs recruitment of effector cells and alters cytokine production, so prolonging graft survival. We also show that low doses of blocking antibodies against leukocyte function associated antigen-1 (LFA-1)/alpha L beta 2 and very late antigen-4 (VLA-4)/alpha 4 beta 1, when combined with deletion of beta 3, lead to long-term survival of allografts with no evidence of chronic rejection. Hence we provide strong mechanistic evidence supporting previous genetic studies, demonstrate the involvement of beta 3 integrins in both acute and chronic rejection and identify beta 3 as a new target for immunosuppressive therapy.

Accelerated re-epithelialization in beta3-integrin-deficient- mice is associated with enhanced TGF-beta1 signaling.

The upregulation of TGF-beta1 and integrin expression during wound healing has implicated these molecules in this process, but their precise regulation and roles remain unclear. Here we report that, notably, mice lacking beta(3)-integrins show enhanced wound healing with re-epithelialization complete several days earlier than in wild-type mice. We show that this effect is the result of an increase in TGF-beta1 and enhanced dermal fibroblast infiltration into wounds of beta(3)-null mice. Specifically, beta(3)-integrin deficiency is associated with elevated TGF-beta receptor I and receptor II expression, reduced Smad3 levels, sustained Smad2 and Smad4 nuclear localization and enhanced TGF-beta1-mediated dermal fibroblast migration. These data indicate that alpha(v)beta(3)-integrin can suppress TGF-beta1-mediated signaling, thereby controlling the rate of wound healing, and highlight a new mechanism for TGF-beta1 regulation by beta(3)-integrins.

Interruption of coding sequences by heterologous introns can enhance the functional expression of recombinant genes.

Sustained expression of recombinant proteins is a critical factor for the effectiveness of numerous applications in the biomedical sciences including the treatment of human disease by gene therapy, the large scale production of therapeutic proteins, as well as the investigation of gene function by transgenesis or cell type specific mutagenesis. Although much attention has been paid to the optimisation of regulatory sequences such as promoters, untranslated regions and polyadenylation signals, effective and sustained expression of recombinant genes in vivo is often difficult to achieve. Here we report that the creation of artificial exons, by insertion of two short heterologous introns into open reading frames, is not only compatible with functional expression, but also leads to a 30-fold enhancement of mRNA production for both green fluorescent protein and the bacteriophage P1-derived Cre recombinase. The levels of green fluorescence were increased five-fold in cell lines and sustained long-term expression at increased levels was observed in rat brain after transduction with a herpes simplex virus-based vector. The data presented identify a means by which the expression of recombinant genes can be enhanced considerably, in addition to and independently from the surrounding regulatory sequences. The method should help obtain sustained and effective expression of recombinant proteins in vivo.

Biological responses to electromagnetic fields.

Electrification in developed countries has progressively increased the mean level of extremely low-frequency electromagnetic fields (ELF-EMFs) to which populations are exposed; these humanmade fields are substantially above the naturally occurring ambient electric and magnetic fields of approximately 10(-4) Vm(-1) and approximately 10(-13) T, respectively. Several epidemiological studies have concluded that ELF-EMFs may be linked to an increased risk of cancer, particularly childhood leukemia. These observations have been reinforced by cellular studies reporting EMF-induced effects on biological systems, most notably on the activity of components of the pathways that regulate cell proliferation. However, the limited number of attempts to directly replicate these experimental findings have been almost uniformly unsuccessful, and no EMF-induced biological response has yet been replicated in independent laboratories. Many of the most well-defined effects have come from gene expression studies; several attempts have been made recently to repeat these key findings. This review analyses these studies and summarizes other reports of major cellular responses to EMFs and the published attempts at replication. The opening sections discuss quantitative aspects of exposure to EMFs and the incidence of cancers that have been correlated with such fields. The concluding section considers the problems that confront research in this area and suggests feasible strategies.

No effect of 60 Hz electromagnetic fields on MYC or beta-actin expression in human leukemic cells.

Epidemiological studies have shown weak correlations between exposure to extremely low-frequency electromagnetic fields (ELF EMFs) and the incidence of several cancers, particularly childhood leukemias, although negative studies have also been reported. These observations have prompted a broad range of in vitro cellular studies in which effects of ELF EMFs have been observed. However, no reported response has been replicated widely in independent laboratories. One potentially important response is the rapid activation of proto-oncogenes and other genes in human leukemic (HL60) cells and a wide variety of other eukaryotic cells, because of the role of these genes in cell proliferation. We describe quantitative Northern analysis of MYC and beta-actin mRNAs from HL60 cells exposed to fields under conditions very similar to those reported previously to activate these genes, namely 60 Hz sinusoidal magnetic fields of 0.57, 5.7 or 57 microT for 20 min. In addition we have used a new design of field-exposure system and introduced a number of other modifications to the protocol to optimize any response. We have also developed a novel method providing enhanced accuracy for the quantitative measurement of mRNA. No significant effect of ELF EMFs on gene expression was observed using any of these systems and analytical methods.