Knockdown of COPA, identified by loss-of-function screen, induces apoptosis and suppresses tumor growth in mesothelioma mouse model.

Malignant mesothelioma is a highly aggressive tumor arising from serosal surfaces of the pleura and is triggered by past exposure to asbestos. Currently, there is no widely accepted treatment for mesothelioma. Development of effective drug treatments for human cancers requires identification of therapeutic molecular targets. We therefore conducted a large-scale functional screening of mesothelioma cells using a genome-wide small interfering RNA library. We determined that knockdown of 39 genes suppressed mesothelioma cell proliferation. At least seven of the 39 genes-COPA, COPB2, EIF3D, POLR2A, PSMA6, RBM8A, and RPL18A-would be involved in anti-apoptotic function. In particular, the COPA protein was highly expressed in some mesothelioma cell lines but not in a pleural mesothelial cell line. COPA knockdown induced apoptosis and suppressed tumor growth in a mesothelioma mouse model. Therefore, COPA may have the potential of a therapeutic target and a new diagnostic marker of mesothelioma.

Comparison of conventional and novel PET tracers for imaging mesothelioma in nude mice with subcutaneous and intrapleural xenografts.

INTRODUCTION: Malignant mesothelioma is a highly aggressive tumor originating in the pleura, peritoneum and pericardium, and the prognosis of patients undergoing current treatment remains poor. To develop new therapies, it is important to have a noninvasive imaging system for evaluating the efficacy of such prospective treatments. We have established clinically relevant mouse models and evaluated conventional and novel positron emission tomography (PET) tracers. METHODS: Epithelioid and sarcomatoid mesothelioma cells were inoculated subcutaneously and intrapleurally into nude mice. Biodistribution and PET imaging studies were conducted by injecting [(18)F]fluoro-2-deoxy-D-glucose (FDG), 3'-[(18)F]fluoro-3'-doxythymidine (FLT) or 4'-methyl-[(11)C]thiothymidine (S-dThd) into the mouse models. In vitro cellular uptake of [(14)C]FDG and [(3)H]FLT and thymidine kinase 1 (TK(1)) activity in both cell lines were measured. Expression of glucose transporter 1 (GLUT-1) and Ki-67 in xenografted tumors was evaluated by immunohistochemical staining. RESULTS: In epithelioid mesothelioma models, biodistribution experiments showed that tumor uptake of [(11)C]S-dThd was significantly higher than that of [(18)F]FDG. On the other hand, in sarcomatoid models, [(18)F]FDG showed significantly higher accumulation than the other two tracers. These differential uptakes of the three tracers were confirmed by PET imaging. The cellular uptake of [(14)C]FDG and [(3)H]FLT and TK(1) activity in sarcomatoid cells were higher than those of epithelioid cells. GLUT-1 protein was strongly expressed in sarcomatoid but not in epithelioid tumor. We observed a high percentage of Ki-67-positive cells in both epithelioid and sarcomatoid tumors. CONCLUSIONS: We established nude mouse models of epithelioid and sarcomatoid subtypes of mesothelioma. PET tracers applicable for the evaluation of epithelioid and sarcomatoid mesothelioma would vary: [(18)F]FLT and [(11)C]S-dThd seemed suitable for the epithelioid subtype and [(18)F]FDG seemed suitable for the sarcomatoid subtype in our mouse models. Our results indicated that cellular uptake and TK(1) activity in vitro are not always consistent with tracer uptake of [(18)F]FLT and [(11)C]S-dThd in vivo. These mouse models and PET imaging might be useful tools for evaluating new and effective treatments in mesothelioma.

A loss of function screen identifies nine new radiation susceptibility genes.

Genomic instability is considered a hallmark of carcinogenesis, and dysfunction of DNA repair and cell cycle regulation in response to DNA damage caused by ionizing radiation are thought to be important factors in the early stages of genomic instability. We performed cell-based functional screening using an RNA interference library targeting 200 genes in human cells. We identified three known and nine new radiation susceptibility genes, eight of which are linked directly or potentially with cell cycle progression. Cell cycle analysis on four of the genes not previously linked to cell cycle progression demonstrated that one, ZDHHC8, was associated with the G2/M checkpoint in response to DNA damage. Further study of the 12 radiation susceptibility genes identified in this screen may help to elucidate the molecular mechanisms of cell cycle progression, DNA repair, cell death, cell growth and genomic instability, and to develop new radiation sensitizing agents for radiotherapy.

Defective repair of radiation-induced DNA damage is complemented by a CHORI-230-65K18 BAC clone on rat chromosome 4.

The Long Evans cinnamon (LEC) rat is highly susceptible to X-irradiation due to defective DNA repair and is thus a model for hepatocellular carcinogenesis. We constructed a bacterial artificial chromosome (BAC) contig of rat chromosome 4 completely covering the region associated with radiation susceptibility. We used transient and stable transfections to demonstrate that defective DNA repair in LEC cells is fully complemented by a 200-kb BAC, CHORI-230-65K18. Further analysis showed that the region associated with radiation susceptibility is located in a 128,543-bp region of 65K18 that includes the known gene Rpn1. However, neither knockdown nor overexpression of Rpn1 indicated that this gene is associated with radiation susceptibility. We also mapped three ESTs (TC523872, TC533727, and CB607546) in the 128,543-bp region, suggesting that 65K18 contains an unknown gene associated with X-ray susceptibility in the LEC rat.

A fast, simple method for screening radiation susceptibility genes by RNA interference.

Radiotherapy can cause unacceptable levels of damage to normal tissues in some cancer patients. To understand the molecular mechanisms underlying radiation-induced physiological responses, and to be able to predict the radiation susceptibility of normal tissues in individual patients, it is important to identify a comprehensive set of genes responsible for radiation susceptibility. We have developed a simple and rapid 96-well screening protocol using cell proliferation assays and RNA interference to identify genes associated with radiation susceptibility. We evaluated the performance of alamarBlue-, BrdU-, and sulforhodamine B-based cell proliferation assays using the 96-well format. Each proliferation assay detected the known radiation susceptibility gene, PRKDC. In a trial screen using 28 shRNA vectors, another known gene, CDKN1A, and one new radiation susceptibility gene, ATP5G3, were identified. Our results indicate that this method may be useful for large-scale screens designed to identify novel radiation susceptibility genes.

Fine mapping of radiation susceptibility and gene expression analysis of LEC congenic rat lines.

LEC rats constitute an animal model of high susceptibility to X-rays. We developed congenic LEC rat lines (recipient strain, Fischer 344 (F344)) and performed genome-wide genotyping to identify radiation susceptibility genes. We mapped seven positional candidate genes, Bmp10, Gpr73, Gp9, Cnbp, Copg, Rab7, and Rpn1, to an approximately 1.2-Mb region located between loci D4Got85 and D4Got148 on chromosome 4. None of the seven genes has been reported to be associated with radiation susceptibility. Comparison of the coding sequences for these seven genes in F344 and LEC rats showed no changes in deduced amino acid sequences. We determined gene expression differences in Gp73, Gp9, and Cnbp as well as strain-specific variations in upstream sequences of these genes. Our results suggest that radiation susceptibility in the LEC rat is primarily attributable to one of the genes within this approximately 1.2-Mb region; however, expression analysis gave no clear indication as to which gene is responsible.

Severe growth retardation and short life span of double-mutant mice lacking Xpa and exon 15 of Xpg.

In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause an early onset of Cockayne syndrome (CS) in some patients (XP-G/CS) with characteristics, such as growth retardation and a short life span. In the previous studies, we generated four Xpg mutant mice with two different C-terminal truncations, null, or a base substitution mutation to identify the protein region that causes the onset of CS, and found that the CS-causing mutations, null or a deletion of the last 360 amino acids, completely inhibited the NER activity of mouse XPG (Xpg), but the non-CS-causing mutations, XpgD811A (base substitution that eliminates the nuclease activity of Xpg) or XpgDeltaex15 (deletion of the exon 15 corresponding to the last 183 amino acids), resulted in the retention of residual NER activity. To understand why mutations that completely eliminate the NER activity of Xpg cause CS but those that abolish the nuclease activity without totally eliminating the NER activity of Xpg do not result in CS, we made a series of Xpg mutant mice with Xpa-null mutant allele and found that mice with the non-CS-causing deletion mutation (XpgDeltaex15) exhibited the CS phenotype when XPA was also absent but the base substitution mutation (XpgD811A) that eliminated the Xpg nuclease activity did not. These results indicate that Xpg has a second function, beside NER, and that the disruption of this second function (deletion of the last 183 amino acids) when combined with an NER defect causes CS. When we compared amino acid sequences corresponding to the exon 15 of Xpg, a significant homology was conserved among vertebrates, but not in Drosophila and Saccharomyces cerevisiae. These observations suggest that the second function of XPG may be conserved only in vertebrates and CS symptoms may occur in its absence.

Identification of the XPG region that causes the onset of Cockayne syndrome by using Xpg mutant mice generated by the cDNA-mediated knock-in method.

In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause early onset of Cockayne syndrome (CS) in some patients (XPG/CS). The CS-causing mutations in such patients all produce truncated XPG proteins. To test the hypothesis that the CS phenotype, with characteristics such as growth retardation and a short life span in XPG/CS patients, results from C-terminal truncations, we constructed mutants with C-terminal truncations in mouse XPG (Xpg) (from residue D811 to the stop codon [XpgD811stop] and deletion of exon 15 [Xpg Delta ex15]). In the XpgD811stop and Xpg Delta ex15 mutations, the last 360 and 183 amino acids of the protein were deleted, respectively. To generate Xpg mutant mice, we devised the shortcut knock-in method by replacing genomic DNA with a mutated cDNA fragment (cDNA-mediated knock in). The control mice, in which one-half of Xpg genomic DNA fragment was replaced with a normal Xpg cDNA fragment, had a normal growth rate, a normal life span, normal sensitivity to UV light, and normal DNA repair ability, indicating that the Xpg gene partially replaced with the normal cDNA fragment retained normal functions. The XpgD811stop homozygous mice exhibited growth retardation and a short life span, but the Xpg Delta ex15 homozygous mice did not, indicating that deletion of the last 360 amino acids results in the CS phenotype but deletion of the last 183 amino acids does not. The XpgD811stop homozygous mice, however, exhibited a slightly milder CS phenotype than did the Xpg null mutant mice, indicating that the XpgD811stop protein still retains some Xpg function that affects the severity of the CS phenotype.

A genetic mouse model carrying the nonfunctional xeroderma pigmentosum group G gene.

A genetic mouse model with a disrupted XPG allele was generated by insertion of neo cassette sequences into exon 3 of the XPG gene by using embryonic stem (ES) cell techniques. The xpg-deficient mice showed distinct developmental characteristics. Their body was marked smaller than that in wild-type littermates since the postnatal day 6, and this postnatal growth failure became more severe with developmental proceeding. Their life span was very short, all of the mutants died by postnatal day 23 after showing great weakness and emaciation. In addition, the mutant homozygous mice also showed some progressive neurological signs, like the lower level of activity and a progressive ataxia. Further examination indicated there was developmental retardation of the brain in the mutant mice. Their brain weight, and thickness of cerebral cortex and cerebellar cortex were significant different from the controls. These characteristics, like small size brain, brain developmental retardation and progressive neurological dysfunctions in the homozygotes were similar to the typical clinical phenotype of the XPG patients and Cockayne syndrome, we believe that the xpgdeficient mice will be an animal model for studying the function of the XP-G protein in nucleotide-excision repair and mechanisms related to the clinic symptoms of XP-G and Cockayne syndrome in humans.