Inhibition of Uracil DNA Glycosylase Alters Frequency and Spectrum of Action-at-a-Distance Mutations Induced by 8-Oxo-7,8-dihydroguanine.

The generation of DNA damage causes mutations and consequently cancer. Reactive oxygen species are important sources of DNA damage and some mutation signatures found in human cancers. 8-Oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the most abundant oxidized bases and induces a G→T transversion mutation at the modified site. The damaged G base also causes untargeted base substitution mutations at the G bases of 5'-GpA-3' dinucleotides (action-at-a-distance mutations) in human cells, and the cytosine deaminase apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) is involved in the mutation process. The deaminated cytosine, i.e., uracil, bases are expected to be removed by uracil DNA glycosylase. Most of the substitution mutations at the G bases of 5'-GpA-3' might be caused by abasic sites formed by the glycosylase. In this study, we expressed the uracil DNA glycosylase inhibitor from Bacillus subtilis bacteriophage PBS2 in human U2OS cells and examined the effects on the GO-induced action-at-a-distance mutations. The inhibition of uracil DNA glycosylase increased the mutation frequency, and in particular, the frequency of G→A transitions. These results indicated that uracil DNA glycosylase, in addition to APOBEC3, is involved in the untargeted mutation process induced by GO.

Neutralization of hepatocyte growth factor leads to retarded cutaneous wound healing associated with decreased neovascularization and granulation tissue formation.

To elucidate biologic functions of hepatocyte growth factor and the c-Met receptor in cutaneous wound healing, we analyzed expression and localization of hepatocyte growth factor and c-Met receptor and used a strategy to neutralize endogenous hepatocyte growth factor in a cutaneous wound healing model in mice. Following excision of full-thickness skin on the dorsum of mice, expression of both hepatocyte growth factor and the c-Met receptor increased transiently in cutaneous tissues. Expressions of hepatocyte growth factor increased as early as 2 d postwounding and reached a peak on day 2, whereas the c-Met receptor expression reached a peak 2-4 d postwounding. Immunolocalization of the c-Met receptor indicated that c-Met receptor expression was upregulated in keratinocytes, vascular endothelial cells, and myofibroblasts in granulation tissue, hence these are potential target cells of hepatocyte growth factor. When normal rabbit IgG or neutralizing anti-hepatocyte growth factor IgG was locally and continuously delivered to subcutaneous lesions, the number of capillary vessels decreased with the neutralization of hepatocyte growth factor and there was an associated decreased expansion of granulation tissue. Likewise, retardation in re-epithelialization and the rate of wound closure occurred with neutralization of endogenous hepatocyte growth factor on days 4 and 7 postwounding. Therefore, hepatocyte growth factor is definitely involved in enhancing cutaneous wound healing processes, including re-epithelialization, neovascularization, and granulation tissue formation.

Recombinant hepatocyte growth factor accelerates cutaneous wound healing in a diabetic mouse model.

We examined effects of recombinant hepatocyte growth factor (HGF) on cutaneous wound healing, using a full-thickness cutaneous excision model in diabetic mice. Topical administration of HGF, as well as basic fibroblast growth factor (bFGF), promoted the rate of wound closure and re-epithelialization. Both HGF and bFGF enhanced expansion of the granulation tissue and stimulated neovascularization on day 7 postwounding, wherein the increase in microvessel density in HGF-treated wounds was higher than that in bFGF-treated wounds. Matrix metalloproteinases (MMP-2 and MMP-9) activities involved in cell migration, angiogenesis, and extracellular matrix (ECM) remodeling, were enhanced by HGF-treatment on day 7. On day 28 postwounding (later stages of wound healing), granulation tissue in bFGF-treated wounds remained to a greater extent than that seen in saline- and HGF-treated wounds. Likewise, bFGF- but not HGF-treatment stimulated DNA synthesis of fibroblasts in granulation tissue, suggesting that HGF stimulates wound healing with lesser degree of susceptibility to cutaneous scarring. We propose that supplement of HGF may be a potential therapeutic approach for treatment of cutaneous ulcer.