MAD2γ, a novel MAD2 isoform,reduces mitotic arrest and is associated with resistance in testicular germ cell tumors

Background: Prolonged mitotic arrest in response to anti-cancer chemotherapeutics, such as DNA-damaging agents, induces apoptosis, mitotic catastrophe, and senescence. Disruptions in mitotic checkpoints contribute resistance to DNA-damaging agents in cancer. MAD2 has been associated with checkpoint failure and chemotherapy response. In this study, a novel splice variant of MAD2, designated MAD2γ, was identified, and its association with the DNA damage response was investigated.

Methods: Endogenous expression of MAD2γ and full-length MAD2 (MAD2α) was measured using RT-PCR in cancer cell lines, normal foreskin fibroblasts, and tumor samples collected from patients with testicular germ cell tumors (TGCTs). A plasmid expressing MAD2γ was transfected into HCT116 cells, and its intracellular localization and checkpoint function were evaluated according to immunofluorescence and mitotic index.

Results: MAD2γ was expressed in several cancer cell lines and non-cancerous fibroblasts. Ectopically expressed MAD2γ localized to the nucleus and reduced the mitotic index, suggesting checkpoint impairment. In patients with TGCTs, the overexpression of endogenous MAD2γ, but not MAD2α, was associated with resistance to cisplatin-based chemotherapy. Likewise, cisplatin induced the overexpression of endogenous MAD2γ, but not MAD2α, in HCT116 cells.

Conclusions: Overexpression of MAD2γ may play a role in checkpoint disruption and is associated with resistance to cisplatin-based chemotherapy in TGCTs.     

MicroRNA‑224 promotes the sensitivity of osteosarcoma cells to cisplatin by targeting Rac1

Osteosarcoma is the most common primary bone tumour in children and adolescents. Accumulating evidence has shown that microRNAs (miRNAs) participate in the development of almost all types of cancer. Here, we investigated the role of miR‐224 in the development and progression of osteosarcoma. We demonstrated that miR‐224 was down‐regulated in osteosarcoma cell lines and tissues. Lower miR‐224 levels were correlated with shorter survivalin osteosarcoma patients. Furthermore, overexpression of miR‐224 suppressed osteosarcoma cell proliferation, migration and invasion and contributed to the increased sensitivity of MG‐63 cells to cisplatin. We identified Rac1 as a direct target gene of miR‐224 in osteosarcoma. Rac1 expression was up‐regulated in the osteosarcoma cell lines and tissues, and there was an inverse correlation between Rac1 and miR‐224 expression in osteosarcoma tissues. Furthermore, rescuing Rac1 expression decreased the sensitivity of miR‐224‐overexpressing MG‐63 cells to cisplatin. We also demonstrated that ectopic expression of Rac1 promoted the proliferation, migration and invasion of miR‐224‐overexpressing MG‐63 cells. These data suggest that miR‐224 plays a tumour suppressor role in the development of osteosarcoma and is related to the sensitivity of osteosarcoma to cisplatin.     

Virulence and pathogenicity of a Candida albicans mutant with reduced filamentation

Deletion of DNA polymerase eta (Rad30/Polη) in pathogenic yeast Candida albicans is known to reduce filamentation induced by serum, ultraviolet, and cisplatin. Because nonfilamentous C. albicans is widely accepted as avirulent form, here we explored the virulence and pathogenicity of a rad30Δ strain of C. albicans in cell‐based and animal systems. Flow cytometry of cocultured fungal and differentiated macrophage cells revealed that comparatively higher percentage of macrophages was associated with the wild‐type than rad30Δ cells. In contrast, higher number of Polη‐deficient C. albicans adhered per macrophage membrane. Imaging flow cytometry showed that the wild‐type C. albicans developed hyphae after phagocytosis that caused necrotic death of macrophages to evade their clearance. Conversely, phagosomes kill the fungal cells as estimated by increased metacaspase activity in wild‐type C. albicans. Despite the morphological differences, both wild‐type and rad30? C. albicans were virulent with a varying degree of pathogenicity in mice models. Notably, mice with Th1 immunity were comparatively less susceptible to systemic fungal infection than Th2 type. Thus, our study clearly suggests that the modes of interaction of morphologically different C. albicans strains with the host immune cells are diverged, and host genetic background and several other attributing factors of the fungus could additionally determine their virulence.     

酸性成纤维细胞生长因子对庆大霉素和顺铂所致肾小管上皮细胞损伤的保护作用

目的:研究酸性成纤维细胞生长因子(aFGF)对庆大霉素(GM)和顺铂(DDP)引起的体外培养肾小管上皮细胞损害的保护作用。方法:大鼠肾皮质经研磨、过网、胰蛋白酶消化,进行肾小管上皮细胞的体外培养。经Cytokeratin 18抗体进行鉴定后,用GM、DDP建立损伤模型,观察aFGF对损伤的肾小管上皮细胞的保护作用。结果:①经形态学观察、GM或DDP组与对照组比较,CAT、SOD、GSH-Px、Na~ -K~ -ATP酶活性下降,而NAG活性和NO、MDA升高(P<0.01或P<0.05),提示肾小管上皮细胞的培养和GM、DDP损伤的建模成功。②aFGF GM或DDP组与GM组或DDP组比较,大多数生化和酶学指标有显著或非常显著性差异(P<0.05或P<0.01);而aFGF GM或DDP组与对照组比较,CAT、NAG、GSH-Px、Na~ -K~ -ATP酶活性无显著性差异,但SOD活性下降、NO、MDA升高尚有显著性差异(P<0.05)。结论:aFGF对GM、DDP损伤的肾小管上皮细胞有明显的保护作用。     

Determination of platinum in rat dorsal root ganglion using ICP-MS

This study evaluated the performance of inductively coupled plasma mass spectrometry for the determination of platinum (Pt) in rat dorsal root ganglion. The method detection limit was found to be 0.008 ng/mL of Pt, which corresponds to 4 pg of Pt per milligram of ganglia. The standard deviations in the tissue matrix were 5.7% or better and minimum matrix effect was observed. Compared to indium, the use of iridium or a combination of iridium and bismuth as internal standard(s) provided more accurate measurement. The Pt in the tissue digestate was stable for a minimum of 46 d at levels above 0.05 ng/mL. Flow injection analysis using undiluted digestates resulted in approximately 20% signal enhancement. Internal standard correction was necessary to obtain accurate results. The method was used in initial studies in which rats were dosed with cisplatin and has shown that Pt accumulates and persists in dorsal rat ganglion following treatment. Paper presented at the Pittsburgh Conference, Atlanta, GA, March 1997     

Cisplatin restores p53 function and enhances the radiosensitivity in HPV16 E6 containing SiHa cells

Most HPV-positive cervical cancer cells possess wild type p53 gene, but its normal p53 functions are disrupted by expression of HPVs E6. Treatment with 0-20 microM cisplatin for 24 h in HPV16 E6 containing SiHa cells suppressed E6 mRNA, reduced E6 protein, and restored p53 expression in dose-dependent manners. Dual-parameter flow cytometric analysis indicated that sub-G(1) apoptotic cells, but not necrotic cells were the major species for cisplatin-induced cytotoxicity in SiHa cells. After 0-10 microM cisplatin treatment, slightly more apoptotic cells appeared from SiHa cells than those from dominant negative p53-transfected SiHa cells. There was no different ionizing radiation (IR)-induced apoptosis in these two different cells. On the other hand, cisplatin enhanced more IR-induced sub-G(1) apoptosis in SiHa than mp53-SiHa cells. These accompanied with prolonged p53 restoration in irradiated-SiHa cells after 24 h cisplatin treatment and thereafter. In contrast, it was not found in cells after irradiation alone. Similar results were also shown in Mdm2 expression in SiHa cells after combined treatment. Therefore, cisplatin restored p53 expression and prolonged IR-induced p53 restoration would be possible candidates to response more sub-G(1) apoptosis in irradiated SiHa cells. These results provided another new explanation on cisplatin sensitizing radiotherapy for HPV16 E6 containing cancer cells.     

IκB/NF-κB mediated cisplatin resistance in HeLa cells after low-dose γ-irradiation is associated with altered SODD expression

Fractionated -irradiation (15 × 2 Gy in 3 weeks) induces a cellular resistance in HeLa cells against cisplatin exposure but not against irradiation. The mechanisms underlying this cellular resistance are associated with major changes in the TNFR1-dependent transduction pathway. The resistant HeLa/B cells exhibit increased levels of NF-B with temporally independent regulation of the subunits NF-B50 and NF-B65. Blocking IB degradation by the proteasome inhibitor PSI, which abolishes the release of the active NF-B protein, induces cell death much more effectively in the parental than in the resistant HeLa/B cells. The translocation of NF-B does not seem to be affected in a similar manner since masking of the translocation sequence by NF-B SN50 enhances cisplatin toxicity to the same degree in both cell lines and overcomes drug resistance. Changes in upstream signaling are suggested by increased sensitivity of the parental HeLa cells to cisplatin in the presence of neutralizing anti-TNFR1. In HeLa/B cells, reduced expression of the 50 kDa silencer of death domain, SODD, is accompanied by constitutive overexpression of a 40–42 kDa SODD-like protein. A possible involvement of SODD in cisplatin resistance is discussed, which may shift the balance between life and death in the TNF receptor pathway to increased NF-B activation.