Role of Atg5-dependent cell death in the embryonic development of Bax/Bak double-knockout mice.

Programmed cell death, which is required for the development and homeostasis of metazoans, includes mechanisms such as apoptosis, autophagic cell death, and necrotic (or type III) death. Members of the Bcl2 family regulate apoptosis, among which Bax and Bak act as a mitochondrial gateway. Although embryonic fibroblasts from Bax/Bak double-knockout (DKO) mice are resistant to apoptosis, we previously demonstrated that these cells die through an autophagy-dependent mechanism in response to various types of cellular stressors. To determine the physiological role of autophagy-dependent cell death, we generated Atg5/Bax/Bak triple-knockout (TKO) mice, in which autophagy is greatly suppressed compared with DKO mice. Embryonic fibroblasts and thymocytes from TKO mice underwent autophagy much less frequently, and their viability was much higher than DKO cells in the presence of certain cellular stressors, providing genetic evidence that DKO cells undergo Atg5-dependent death. Compared with wild-type embryos, the loss of interdigital webs was significantly delayed in DKO embryos and was even further delayed in TKO embryos. Brain malformation is a distinct feature observed in DKO embryos on the 129 genetic background, but not in those on a B6 background, whereas such malformations appeared in TKO embryos even on a B6 background. Taken together, our data suggest that Atg5-dependent cell death contributes to the embryonic development of DKO mice, implying that autophagy compensates for the deficiency in apoptosis.

Combination of HDAC inhibitor MS-275 and IL-2 increased anti-tumor effect in a melanoma model via activated cytotoxic T cells.

BACKGROUND: Histone deacetylase (HDAC) inhibitors are immunomodulatory, and demonstrate antitumor activity in various tumor models including malignant melanoma. OBJECTIVE: The present study examines the effectiveness of IL-2 and HDAC inhibitor MS-275-combination therapy in a murine melanoma model. METHODS: B16F10 cells were implanted subcutaneously in C57BL/6 mice which were randomly divided into four groups and treated with either IL-2 by subcutaneous injection, MS-275 by oral gavage (5 days/week, daily for 2 weeks), or a combination of the two agents. RESULTS: MS-275 treatment showed a dose-dependent inhibitory effect on B16 cells in a colonogenic assay. Flow cytometry analysis indicated that MS-275 induced G1 arrest but not apoptosis in vitro, but IL-2 failed to inhibit cell proliferation. The combination of MS-275 and IL-2 had a statistically significant additive inhibitory effect on melanoma tumor weight and volume in vivo. Significantly higher survival was evident in the combination group compared with the control or single-agent groups. The combination therapy produced a greater ratio of CD8(+) CD69(+) T cells in lymph nodes than was seen in the MS-275-treatment and no-treatment groups among tumoriferous mice. Splenocytes from mice treated with MS-275 and the combination therapy demonstrated greater lysis of melanoma cells in vitro than splenocytes from mice treated with IL-2 or those without treatment. A significant antitumor effect from IL-2 and MS-275-combination therapy in vivo was seen in the increased number of activated CD8(+) T cells. CONCLUSIONS: These data provide a convincing rationale for considering the role of epigenetics in future treatments for malignant melanoma.

Inhibition of epithelial cell death by Bcl-2 improved chronic colitis in IL-10 KO mice.

IL-10-deficient mice spontaneously develop intestinal inflammation, which has many similarities to Crohn's disease. Several reports suggest that epithelial cell death may increase the severity of colitis; however, decisive evidence is lacking. In the present report, we addressed whether and how epithelial cell death plays a role in the development of chronic colitis. We first examined the morphological characteristics of intestines of IL-10-deficient mice and found two forms of epithelial cell death (typical apoptosis and necrosis-like cell death) in colitis. To elucidate the pathological roles of epithelial cell death, we crossbred IL-10-deficient knockout mice with Bcl-2 transgenic mice, in which the anti-apoptosis protein Bcl-2 was overexpressed in intestinal epithelial cells, but not in immune cells. Epithelial cell-specific Bcl-2 protected IL-10 deficiency-induced colitis and markedly reduced their symptoms. Interestingly, morphological analysis revealed that Bcl-2 suppressed apoptosis and necrosis-like cell death, and better maintained mucosal barrier in IL-10-deficient mice. From the immunological aspect, Bcl-2 did not alter the activation of T-helper cell 1 but inhibited the growth of T-helper cell 17, suggesting that mucosal integrity may control the immune responses. These results provide genetic evidence demonstrating that epithelial cell death is crucial for the development of chronic colitis.

N-cadherin expression is a potential survival mechanism of gefitinib-resistant lung cancer cells.

Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer and is the most common and fatal cancer worldwide. Specific tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR), such as gefitinib, have been effective in some NSCLC patients and are being used in the clinical setting as pioneer molecularly targeted cancer drugs. However, many patients have not responded to these drugs, and have acquired resistance after long-term treatment. To identify other potential NSCLC molecular targets, we used DNA microarrays to examine gene expression profiles of gefitinib-resistant PC9/ZD cells that are derived from gefitinib-sensitive PC9 cells and harbor a threonine to methionine mutation at codon 790 (T790M) in EGFR, a known mechanism of acquired resistance to gefitinib. We found that N-cadherin expression was significantly upregulated in PC9/ZD cells compared with PC9 cells. Inhibition of N-cadherin expression by siRNA or treatment with antibodies against N-cadherin induced apoptosis of PC9/ZD cells in association with reduced phosphorylation of Akt and Bad, a proapoptotic protein. Moreover, inhibition of Akt expression by siRNA or treatment with an inhibitor for phosphatidylinositol (PI)-3 kinase reduced survival of PC9/ZD cells. In addition, we found several N-cadherin-expressing lung cancer cells that showed inherent resistance to gefitinib treatment and reduced survival owing to siRNA-induced inhibition of N-cadherin expression. Thus, it appears that N-cadherin maintains the survival of the gefitinib-resistant lung cancer cells via the PI-3 kinase/Akt survival pathway. From these results, we propose that N-cadherin signaling contributes, at least in part, to the survival mechanisms of gefitinib-resistant NSCLC cells and that N-cadherin is a potential molecular target in the treatment of NSCLC.

Hair growth stimulatory effect by a combination of 5-aminolevulinic acid and iron ion.

BACKGROUND: 5-Aminolevulinic acid (5-ALA) is a precursor of a tetrapyrrole compound. 5-ALA has been used for photodynamic therapy as well as for plant growth. 5-ALA and iron ion are precursors of heme, which is incorporated into hemoglobin and cytochrome. AIM: To explore the possible application of a 5-ALA and iron ion admixture on hair growth in mice. METHODS: The effect of a 5-ALA and iron ion admixture on hair growth and cell proliferation in mice was examined. The dorsal hair of 8-week-old male CeH/HeN mice was clipped, and a 5-ALA and iron ion admixture was applied to the dorsal skin once daily for 21 days in a room supplied with common room lights. Hair growth was later examined by calculating the ratio of the area showing hair growth to the total clipped area. For the cell proliferation assay, a 5-ALA and iron ion admixture at several different concentrations was added to a culture medium containing keratinocytes or fibroblasts, and the cell numbers were counted. RESULTS: Mice treated with an admixture of 5-ALA and iron ion showed significant hair growth (P < 0.05) at day 15 relative to those treated with iron ion alone, as revealed by the Tukey-Kramer test. The stimulatory effect of the mixture was almost identical to that of 5% minoxidil. No proliferation of keratinocytes or fibroblasts was observed, however, when an admixture of 5-ALA and iron ion was added to the medium. CONCLUSIONS: The results suggest that an admixture of 5-ALA and iron ion stimulates murine hair growth in vivo independent of epithelial and mesenchymal cells, although the precise mechanism is still uncertain. This mixture has the potential to become a beneficial new treatment for alopecia.