BackgroundResistance to chemotherapy is a major obstacle in the effective treatment of cancer patients. B7-homolog 1, also known as programmed death ligand-1 (PD-L1), is an immunoregulatory protein that is overexpressed in several human cancers. Interaction of B7-H1 with programmed death 1 (PD-1) prevents T-cell activation and proliferation, sequestering the T-cell receptor from the cell membrane, inducing T-cell apoptosis, thereby leading to cancer immunoresistance. B7-H1 upregulation contributes to chemoresistance in several types of cancer, but little is known with respect to changes associated with 5-fluorouracil (5-FU) or gastrointestinal cancers.MethodsHCT 116 p53+/+, HCT 116 p53−/− colorectal cancer (CRC) and OE33 esophageal adenocarcinoma (EAC) cells were treated with increasing doses of 5-FU (0.5 uM, 5 uM, 50 uM, 500 uM) or interferon gamma (IFN-γ, 10 ng/mL) in culture for 24 h and B7-H1 expression was quantified using flow cytometry and western blot analysis. We also evaluated B7-H1 expression, by immunohistochemistry, in tissue collected prior to and following neoadjuvant therapy in 10 EAC patients.ResultsB7-H1 expression in human HCT 116 p53+/+ and HCT 116 p53−/− CRC cells lines, while low at baseline, can be induced by treatment with 5-FU. OE33 baseline B7-H1 expression exceeded CRC cell maximal expression and could be further increased in a dose dependent manner following 5-FU treatment in the absence of immune cells. We further demonstrate tumor B7-H1 expression in esophageal adenocarcinoma patient-derived pre-treatment biopsies. While B7-H1 expression was not enhanced in post-treatment esophagectomy specimens, this may be due to the limits of immunohistochemical quantification.ConclusionsB7-H1/PD-L1 expression can be increased following treatment with 5-FU in gastrointestinal cancer cell lines, suggesting alternative mechanisms to classic immune-mediated upregulation. This suggests that combining 5-FU treatment with PD-1/B7-H1 blockade may improve treatment in patients with gastrointestinal adenocarcinoma.
Bardet–Biedl syndrome (BBS) is a rare, primarily autosomal-recessive ciliopathy. The phenotype of this pleiotropic disease includes retinitis pigmentosa, postaxial polydactyly, truncal obesity, learning disabilities, hypogonadism and renal anomalies, among others. To date, mutations in 15 genes (BBS1–BBS14, SDCCAG8) have been described to cause BBS. The broad genetic locus heterogeneity renders mutation screening time-consuming and expensive. We applied a strategy of DNA pooling and subsequent massively parallel resequencing (MPR) to screen individuals affected with BBS from 105 families for mutations in 12 known BBS genes. DNA was pooled in 5 pools of 21 individuals each. All 132 coding exons of BBS1–BBS12 were amplified by conventional PCR. Subsequent MPR was performed on an Illumina Genome Analyzer II™ platform. Following mutation identification, the mutation carrier was assigned by CEL I endonuclease heteroduplex screening and confirmed by Sanger sequencing. In 29 out of 105 individuals (28%), both mutated alleles were identified in 10 different BBS genes. A total of 35 different disease-causing mutations were confirmed, of which 18 mutations were novel. In 12 additional families, a total of 12 different single heterozygous changes of uncertain pathogenicity were found. Thus, DNA pooling combined with MPR offers a valuable strategy for mutation analysis of large patient cohorts, especially in genetically heterogeneous diseases such as BBS.
The purpose of this study was to determine the role of the retinol dehydrogenase 12 (RDH12) gene in patients affected with Leber congenital amaurosis (LCA), autosomal recessive retinitis pigmentosa (arRP) and autosomal dominant/recessive cone-rod dystrophies (CORD). Changes in the promoter region, coding regions and exon/intron junctions of the RDH12 gene were evaluated using direct DNA sequencing of patients affected with LCA (n=36 cases), RP (n=62) and CORD (n=21). The allele frequency of changes observed was assessed in a multiethnic control population (n=159 individuals). Detailed biochemical and structural modeling analysis of the observed mutations were performed to assess their biological role in the inactivation of Rdh12. A comprehensive clinical assessment of retinal structure and function in LCA patients carrying mutations in the RDH12 gene was completed. Of the six changes identified, three were novel including a homozygous C201R change in a patient affected with LCA, a heterozygous A177V change in patients affected with CORD and a heterozygous G46G change in a patient affected with LCA. A novel compound heterozygote T49M/A269fsX270 mutation was also found in a patient with LCA, and both homozygous and heterozygous R161Q changes were seen in 26 patients affected with LCA, CORD or RP. These R161Q, G46G and the A177V sequence changes were shown to be polymorphic. We found that Rdh12 mutant proteins associated with LCA were inactive or displayed only residual activity when expressed in COS-7 and Sf9 cells, whereas those mutants that were considered polymorphisms were fully active. Thus, impairment of retinal structure and function for patients carrying these mutations correlated with the biochemical properties of the mutants.
Checkpoint inhibitors (eg, programmed cell death protein 1 [PD-1], programmed cell death ligand 1 [PD-L1], cytotoxic T-lymphocyte associated protein 4 [CTLA-4] antibodies) are changing how we understand cancer and provide a means to develop modern immunotherapies. An emergent notion relates success with checkpoint inhibitors with high mutational load tumors. There are few studies that examine checkpoint protein expression and relate these to clinical outcomes after the conventional treatment of patients with esophageal cancer, which has a high mutational load. The objective of this review is to summarize the literature that examines checkpoint expression and clinical outcomes, as well as propose an accelerated approach to introducing these therapies into the clinic to treat patients with esophageal cancer.
Treatment of mice with the carcinogen azoxymethane (AOM) induces a number of lesions in the colon, including hyperplastic lesions, as well adenomas and carcinomas in situ. Inbred strains of mice show different responses to AOM-induced carcinogenesis. A/J mice are highly susceptible and develop a greater number of hyperplastic lesions and tumors (15-70 tumors per mouse) than resistant C57BL/6J mice (0-6 tumors per mouse). Susceptibility to AOM-induced tumors segregates as a co-dominant trait in (A Â B6)F1 hybrids. Using a set of 23 AcB and BcA recombinant congenic mouse strains derived from A/J (susceptible) and B6 (resistant) parents, we observed that the number of hyperplastic lesions and tumors induced by AOM was under different genetic controls in AcB/BcA strains. The multiplicity of AOM-induced tumors is controlled by a major locus that we have mapped on the distal portion of chromosome 3, to which we have given the temporary designation colon cancer susceptibility locus 3 (Ccs3). B6 and A/J alleles at Ccs3 are associated with resistance and susceptibility, respectively. Haplotype analysis in key informative AcB/BcA strains restricts the size of the Ccs3 locus to a 14 Mb segment that contains 94 annotated genes. The expression level of all these genes in normal colon has been established by transcript profiling with microarrays, and has led to the identification of a subset of positional candidates that are expressed at high levels in this tissue. The 4q and 1p human chromosomal segments sharing syntenic homology with the mouse Ccs3 segment are known to be associated with inflammatory bowel diseases and colorectal tumors in humans, suggesting that the study of the mouse Ccs3 locus may help further the pathogenesis of these human conditions.
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