Characterization of mechanism involved in acquired resistance to sorafenib in a mouse renal cell cancer RenCa model

PURPOSE: The objective of this study was to investigate the mechanism mediating the acquisition of a resistant phenotype to sorafenib in renal cell carcinoma (RCC). METHODS: A parental mouse RCC cell line, RenCa (RenCa/P), was continuously exposed to increasing doses of sorafenib, and a cell line resistant to sorafenib (RenCa/R), showing an approximately sixfold higher IC50 than that of RenCa/P, was established. Changes in the expression of several molecules in these cell lines following sorafenib treatment were evaluated by western blotting, and the effects of sorafenib treatment on the in vivo growth patterns were compared. RESULTS: There were no significant differences in sensitivities to potential agents against RCC between RenCa/P and RenCa/R. Among several apoptosis-related proteins, the expression of clusterin in RenCa/R was significantly greater than that in RenCa/P. Following treatment with sorafenib, the expression level of phosphorylated p44/42 mitogen-activated protein kinase (MAPK) in RenCa/P, but not that in RenCa/R, was significantly decreased. Furthermore, additional treatment with a specific inhibitor of the MAPK signaling pathway significantly increased the sensitivity of RenCa/R to sorafenib, but not that of RenCa/P. There was no significant difference between the in vivo growth patterns of RenCa/P and RenCa/R in mice without sorafenib treatment; however, the growth inhibitory effect of sorafenib on the RenCa/P tumor was significantly greater than that on the RenCa/R tumor. CONCLUSIONS: These findings suggest that the upregulation of clusterin and continuous activation of the MAPK pathway during sorafenib treatment may be involved in the acquisition of a resistance to sorafenib in RCC (AU)

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Characterization of mechanism involved in acquired resistance to sorafenib in a mouse renal cell cancer RenCa model.

PURPOSE: The objective of this study was to investigate the mechanism mediating the acquisition of a resistant phenotype to sorafenib in renal cell carcinoma (RCC). METHODS: A parental mouse RCC cell line, RenCa (RenCa/P), was continuously exposed to increasing doses of sorafenib, and a cell line resistant to sorafenib (RenCa/R), showing an approximately sixfold higher IC(50) than that of RenCa/P, was established. Changes in the expression of several molecules in these cell lines following sorafenib treatment were evaluated by western blotting, and the effects of sorafenib treatment on the in vivo growth patterns were compared. RESULTS: There were no significant differences in sensitivities to potential agents against RCC between RenCa/P and RenCa/R. Among several apoptosis-related proteins, the expression of clusterin in RenCa/R was significantly greater than that in RenCa/P. Following treatment with sorafenib, the expression level of phosphorylated p44/42 mitogen-activated protein kinase (MAPK) in RenCa/P, but not that in RenCa/R, was significantly decreased. Furthermore, additional treatment with a specific inhibitor of the MAPK signaling pathway significantly increased the sensitivity of RenCa/R to sorafenib, but not that of RenCa/P. There was no significant difference between the in vivo growth patterns of RenCa/P and RenCa/R in mice without sorafenib treatment; however, the growth inhibitory effect of sorafenib on the RenCa/P tumor was significantly greater than that on the RenCa/R tumor. CONCLUSIONS: These findings suggest that the upregulation of clusterin and continuous activation of the MAPK pathway during sorafenib treatment may be involved in the acquisition of a resistance to sorafenib in RCC.

IKBL promoter polymorphism is strongly associated with resistance to type 1 diabetes in Japanese.

Type 1 diabetes is a multifactorial disease in which the genes of the major histocompatibility complex (MHC) play a key role. Recently, non-human leukocyte antigen (non-HLA) genes in the class III region of this complex have been presumed to be associated with type 1 diabetes by linkage analyses. We investigated the possibility of the inhibitor of kappaB-like (IKBL, also known as 'NFKBIL1') gene as one of these candidates. We carried out a case-control study of 124 patients with type 1 diabetes and 330 healthy control subjects. The haplotypes of the IKBL promoter, i.e., PA (-263A, -63T), PB (-263A, -63A), PC (-263G, -63T), were assigned by the single-nucleotide polymorphisms at positions -263 and -63 from the transcription start site. The frequency of the wild-type haplotype, PA, was elevated, while that of the variant-type haplotype, PC, was lower in patients than controls. In two-locus analyses with HLA-DRB1 alleles, the PA haplotype showed linkage disequilibrium with the DRB1*0405 allele and the PC haplotype with the DRB1*1502 allele. A notable observation was that the PC haplotype was significantly associated with protection in the DRB1*1502-negative population. Our study indicates the first evidence of a possible independent association between type 1 diabetes and polymorphisms in the promoter of the IKBL gene.

Hb Tsukumi [beta117(G19)His-->Tyr]: a new hemoglobin variant found in a Japanese male.

We accidentally observed an abnormal elution pattern on high performance liquid chromatogram when we examined the Hb A1c level in a 65-year-old male patient who suffered from pneumoconiosis and alcoholic liver injury. The value of the glycated fraction was within the normal range but the elution patterns on high performance liquid chromatography varied with the glycohemoglobin analyzers. Isoelectrofocusing and urea-cellulose column chromatography showed an anomalous fast-moving beta chain estimated at approximately 47%. The instability test of the hemolysate was slightly positive. Structural analysis demonstrated that the mutant was consisted by a substitution of His-Tyr at beta117. This new variant was named Hb Tsukumi for the place of residence of the patient. Additionally, the nucleotide sequence showed a change of C-->T [CAC (His)-->TAC (Tyr)] at the first base in the 117th codon of the beta gene.

Analysis of tumor necrosis factor-alpha promoter polymorphism in type 1 diabetes: HLA-B and -DRB1 alleles are primarily associated with the disease in Japanese.

Polymorphisms in the 5'-flanking region of the tumor necrosis factor (TNF)-alpha gene were examined to study the genetic background of type 1 diabetes in Japanese. Five different biallelic polymorphisms were examined in 136 type 1 diabetic patients and 300 control subjects. The frequencies of individuals carrying TNF-alpha-857T allele (designated as TNFP-D allele) or -863A/-1,031C allele (designated as TNFP-B allele) were significantly increased in the patients as compared with the controls. Since these TNF-alpha alleles are in linkage disequilibria with certain DRB1 and HLA-B alleles, two-locus analyses were carried out. The TNFP-D allele did not increase the risk in either the presence or absence of the DRB1*0405 or HLA-B54 allele, while the DRB1*0405 and HLA-B54 alleles per se could confer susceptibility in both the TNFP-D allele-positive and -negative populations. Moreover, an odds ratio was remarkably elevated in the population carrying both DRB1*0405 and HLA-B54. Similarly, the TNFP-B allele did not show significant association with the disease in either the HLA-B61-positive or -negative population, while the HLA-B61 allele could significantly increase the risk in the TNFP-B allele-positive population. These data suggest that the associations of TNFP-D and -B alleles may be secondary to their linkage disequilibria with the susceptible HLA class I and class II alleles. Because HLA-B and DRB1 genes were independently associated, both of these genes may be contributed primarily to the pathogenesis of type 1 diabetes in Japanese.

Novel mutations of the ATP7B gene in Japanese patients with Wilson disease.

Wilson disease (WD) is an autosomal recessive disorder characterized by copper accumulation in the liver, brain, kidneys, and corneas, and culminating in copper toxication in these organs. In this study, we analyzed mutations of the responsible gene, ATP7B, in four Japanese patients with WD. By direct sequencing, we identified five mutations, of which two were novel, and 16 polymorphisms, of which 6 were novel. The mutations 2871delC and 2513delA shift the reading frame so that truncated abnormal protein is expected. In contrast to these mutations found in patients with hepatic-type of early onset, the mutations A874V, R778L, and 3892delGTC were either missense mutations or in frame 1-amino acid deletion, and occurred in the patients with hepato-neurologic type of late onset. The mutations 2871delC and R778L have been previously reported in a relatively large number of Japanese patients. In particular, R778L is known to be more prevalent in Asian countries than in other countries of the world. Our data are compatible with the hypothesis that the mutations tend to occur in a population-specific manner. Therefore, the accumulation of the types of mutations in Japanese patients with WD will facilitate the fast and effective genetic diagnosis of WD in Japanese patients.

Novel germline mutations of the MEN1 gene in Japanese patients with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroid glands, the pancreatic islet cells, and the anterior pituitary. Germline mutations of the MEN1 gene in three independent Japanese cases with MEN1 were analyzed. Case 1 has revealed a 2-bp (TA) insertion at nucleotide position 341 (341insTA) in exon 2, which shifts the reading frame such that the mutant protein has a completely different amino acid sequence from codon 78 to the premature stop codon at 119. In case 2, a nucleotide substitution, i.e., TAG in place of TGG, which encodes tryptophan at codon 198 was identified (nonsense mutation). These mutations were heterozygously present and have not been reported previously. Case 3 showed no mutations in the protein-coding exons and exon-intron junctions of the MEN1 gene by single-strand conformation polymorphism or direct sequencing of the polymerase chain reaction (PCR) fragments. We confirmed the finding that patients with MEN1 carry heterozygous germline mutations in the MEN1 gene, which is compatible with the idea that the MEN1 gene is a tumor suppressor gene. The reason why mutations in the coding region of the MEN1 gene could not be detected by PCR-based analysis in some of the MEN1 patients, e.g. case 3, needs to be clarified further.

[Metastatic lung tumor of a thyroid cancer origin detected by a periodic health examination--a case of a complete response].

Reports is the case of a 21-year-old student, who received a medical examination at our Department for an abnormal chest shadow that had been detected during a periodic health examination at his university. No special subjective symptoms such as a cough and/or sputa, pyrexia, pectoralgia, and dyspnea were noted. A thyroid tumor was palpable and a 99mTc thyroid scintigram and a 201Tl thyroid tumor scintigram gave cause to suspect thyroid cancer and a metastatic lung tumor. A total thyroidectomy and a right modified neck dissection revealed a tumor (histologically, a papillary cancer), 3.5 cm in diameter, that mainly involved the right lobe. It was found that 131I-100 mCi internal irradiation was very effective for the metastatic lung tumor, and no abnormal shadow was subsequently revealed by chest X-ray. Now, 6 years later, he is alive with no manifestations of a local relapse nor any abnormal chest X-ray findings.