Targeting protein-trafficking pathways alters melanoma treatment sensitivity

Protein-trafficking pathways are targeted here in human melanoma cells using methods independent of oncogene mutational status, and the ability to up-regulate and down-regulate tumor treatment sensitivity is demonstrated. Sensitivity of melanoma cells to cis-diaminedichloroplatinum II (cDDP, cis-platin), carboplatin, dacarbazine, or temozolomide together with velaparib, an inhibitor of poly (ADP ribose) polymerase 1, is increased by up to 10-fold by targeting genes that regulate both protein trafficking and the formation of melanosomes, intracellular organelles unique to melanocytes and melanoma cells. Melanoma cells depleted of either of the protein-trafficking regulators vacuolar protein sorting 33A protein (VPS33A) or cappuccino protein (CNO) have increased nuclear localization of cDDP, increased nuclear DNA damage by platination, and increased apoptosis, resulting in increased treatment sensitivity. Depleted cells also exhibit a decreased proportion of intracellular, mature melanosomes compared with undepleted cells. Modulation of protein trafficking via cell-surface signaling by binding the melanocortin 1 receptor with the antagonist agouti-signaling protein decreased the proportion of mature melanosomes formed and increased cDDP sensitivity, whereas receptor binding with the agonist melanocyte-stimulating hormone resulted in an increased proportion of mature melanosomes formed and in decreased sensitivity (i.e., increased resistance) to cDDP. Mutation of the protein-trafficking gene Hps6, known to impair the formation of mature melanosomes, also increased cDDP sensitivity. Together, these results indicate that targeting protein-trafficking molecules markedly increases melanoma treatment sensitivity and influences the degree of melanosomes available for sequestration of therapeutic agents.     

From the Cover: Conditional control of gene function by an invertible gene trap in zebrafish

Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.High throughput functional genomic and informatic methods have been developed to interrogate the genome and extract functional predictions about many genes at a time. However, careful phenotypic analysis of genetic mutants remains the sine qua non of reductionist biological science. In most experimental organisms, random mutagenesis is the preferred or only mutagenic technique available. DNA alkylating agents, transposable elements, or retroviruses are traditionally used in these organisms. A major limitation of these traditional genetic methods is that they reveal only the earliest and/or most prominent function of a gene as later functions are masked by the earlier phenotype, which is often lethality. To assess later functions, for example in metabolism, aging, or behavior, conditional alleles are required.The development of conditional alleles has proven a boon to studying gene function in temporally or spatially restricted contexts. Traditional conditional alleles disrupt gene function by changing the environment, for example by increasing the temperature. Engineered conditional alleles disrupt gene function by activating a recombination-mediated molecular switch that ablates gene function in one state, but has no functional consequences in the other state (1, 2). In the mouse, engineered conditional alleles can be generated by homologous recombination to insert the molecular switch at defined loci or by retroviral-mediated random insertion of the molecular switch (3, 4). The second approach leverages the orientation-dependent gene disruption of a gene trap and the ability of Flp/Cre recombinases to stably invert the gene trap. By strategically arranging dimers of heterotypical flp- and cre-recombinase binding sites flanking the gene trap, stable inversion is achieved in cis by recombinase-mediated Flip and Excision (FlEx) (5). However, this conditional gene-trap mutagen has not been validated at the organismal level.A distinct advantage of FlEx-based conditional gene-trap mutations is the possibility of stage- and tissue-specific rescue or knockout of the mutated genes. In zebrafish, several gene-trap mutagenesis methods have been developed (6, 7), including the “gene-break” (6, 8) and “FlipTrap” (9) technologies. We set out to test whether the FlEx-based conditional gene-trap mutagenesis approach functions at the organismal level in zebrafish. We show here that a highly mutagenic transposable element can be used for conditional analysis of essential genes.     

牙龈卟啉单胞菌PG0839基因突变株的构建

目的构建牙龈卟啉单胞菌毒力岛基因PG0839突变菌株,为研究PG0839基因功能提供实验基础。方法扩增1 584 bp PG0839基因片段,对聚合酶链反应（PCR）产物和pUC19载体进行BamH Ⅰ和EcoRⅠ双酶切,连接酶切产物得到质粒pPG0839-1。将2 101 bp erm基因产物插入到pPG0839-1中PG0839基因的EcoRⅤ位点,构建质粒pPG0839-2,作为电穿孔的供体质粒。电穿孔转化于受体菌牙龈卟啉单胞菌W83菌株,红霉素抗性培养基筛选阳性克隆,命名为PG0839基因突变菌株。结果运用插入失活方法构建PG0839基因突变菌株,进而通过酶切、测序、PR和反转录PCR对PG0839基因突变菌株进行验证,证实PG0839基因突变菌株构建成功。结论本实验成功构建PG0839基因突变菌株。     

Areal measures of healthy adults' hippocampal formation on brain magnetic resonance imaging

This study measured the area of the hippocampal structure using brain magnetic resonance imaging. We rebuilt a three-dimensional reconstruction of the brain and selected specific sections on the coronal plane, the cross section, and the sagittal plane and then measured the areas of the hippocampus using a software on the computer. In different sections, the left and right hippocampal areas were significantly different (P < 0.05), but the hippocampal areas of males and females are not concordant. There is no significant difference in the area in those aged 20 to 60 years. The hippocampal area is not correlated with the brain area in the same section. In conclusion, the standardization of the hippocampus is not suitable to judge the difference between males and females. The hippocampal area of healthy adults provides the criteria for atrophy of the hippocampus and a brief approach instead of the volumetric measures to apply in clinical diagnosis.     

化疗前后Caspase-3和P73在颊粘膜鳞状细胞癌中的表达及其临床意义

目的研究Caspase-3和P73在颊粘膜鳞状细胞癌中应用奥铂联合替加氟化疗前后的表达及临床意义。方法选取30例颊粘膜鳞状细胞癌患者的癌组织为实验组,10例颊粘膜良性肿瘤患者的正常黏膜为对照组。将癌组织和正常粘膜组织分为三组,即A组(化疗前)、B组(化疗后)和C组(正常粘膜)。应用免疫组织化学SP法和流式细胞术,检测Caspase-3和P73在各组中的表达。结果①免疫组化:Caspase-3在化疗前颊粘膜鳞状细胞癌中阳性率为69.65±2.75%,化疗后为83.96±3.37%,正常黏膜组织阳性率为98.53±1.08%;P73在化疗前颊粘膜鳞状细胞癌中阳性率为89.57±4.0%,化疗后为71.19±12.3%,正常黏膜组织阳性率为54.85±4.43%。②流式细胞术:Caspase-3在化疗前颊粘膜鳞状细胞癌中的表达为0.715±0.063,化疗后为0.880±0.038,正常口腔粘膜细胞为1.000±0.091。各组之间比较有显著性差异(P<0.05)。P73在化疗前为1.310±0.065,化疗后为1.134±0.037,正常粘膜细胞为1.000±0.091。各组之间比较有显著性差异(P<0.05)。结论 Caspase-3和P73可能是颊黏膜癌变的早期事件,可作为癌变监测和临床疗效观察的辅助指标。